Synergistic combination of immunologic inhibitors for the treatment of cancer

ABSTRACT

The present disclosure provides combinations of immunologic inhibitors for the treatment of cancer. In some embodiments, the methods involve the use of a combination of at least two of the following: an inhibitor of indoleamine-2,3-dioxygenase (IDO), an inhibitor of the PD-L1/PD-1 pathway, an inhibitor of CTLA-4, an inhibitor of CD25, or IL-7.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.14/438,310 filed Apr. 24, 2015, which is a national phase applicationunder 35 U.S.C. § 371 of International Application No. PCT/US2013/066936filed Oct. 25, 2013, which claims the benefit of priority to U.S.Provisional Patent Application Ser. No. 61/719,202, filed Oct. 26, 2012.The entire contents of each of the above-referenced disclosures arespecifically incorporated herein by reference.

BACKGROUND OF THE INVENTION I. Field of the Invention

Embodiments of this invention are directed generally to microbiology andmedicine. In certain aspects the invention is directed to treatment ofcancer.

II. Description of the Related Art

Over the last two decades, numerous mechanisms have been identified thatcontribute to immune suppression in the context of a growing tumor.Recent work has suggested that failed immune-mediated tumor rejection isattributable to the dominant inhibitory effect of immune suppressivemechanisms that largely act at the level of the tumor microenvironment.At least 4 such mechanisms appear to be operative in individual tumors:ligation of PD-1 on T cells by PD-L1 expressed by tumor cells, extrinsicsuppression by regulatory T cells (Tregs), classical T cell anergy, andtryptophan catabolism by indoleamine-2,3-dioxygenase (IDO). Preclinicalstudies have revealed that manipulation each of these pathwaysindividually can have a positive effect on anti-tumor immunity.

Data also suggest that Treg cells may serve as an important therapeutictarget for patients with early stages of cancer and that more vigorouscombinatorial approaches simultaneously targeting multiple immuneevasion as well as immunosurveillance mechanisms for the generation of aproductive immune response against tumor may be required for effectiveimmunotherapy in patients with advanced disease (Elpek et al., 2007).

The first therapeutic approach to reach clinical practice is theblockade of CTLA-4 using the anti-CTLA-4 mAb ipilimumab. Despite thistherapeutic advance, this regimen is effective in only a minority ofpatients, and therefore further treatment developments are required.

Preclinical studies have revealed that manipulation each of thesepathways individually can have a positive effect on anti-tumor immunity.However, as the tumor microenvironment appears to involve the complexinterplay between multiple pathways together, it may be necessary toblock 2 or more immune suppressive mechanisms in concert in order toachieve a maximal therapeutic effect. In addition, otherimmunomodulatory pathways appear to be operational outside the tumormicroenvironment in secondary lymphoid structures—one such regulatorymolecule is CTLA-4, and the anti-CTLA-4 mAb ipilimumab was recentlyapproved for treatment of patients with metastatic melanoma.Simultaneous manipulation of pairs of immune regulatory pathwaystogether may yield synergistic effects on the anti-tumor immuneresponse, thus translating into improved tumor control in vivo.

SUMMARY OF THE INVENTION

In some aspects, there are methods of treating cancer in a subjectcomprising administering to the subject an effective amount of at leasttwo of the following: an inhibitor of indoleamine-2,3-dioxygenase (IDO),an inhibitor of the PD-L1/PD-1 pathway, an inhibitor of CTLA-4, aninhibitor of CD25, or IL-7. Any combination of at least two of thefollowing: an inhibitor of indoleamine-2,3-dioxygenase (IDO), aninhibitor of the PD-L1/PD-1 pathway, an inhibitor of CTLA-4, aninhibitor of CD25, or IL-7 may be used. In some embodiments, the methodmay comprise the use of two or more inhibitors. In some embodiments, themethod may comprise the use of three or more inhibitors. In someembodiments, the method may comprise the use of four or more inhibitors.In some embodiments, the method may comprise the use of five or moreinhibitors. In some embodiments, the method comprises administering aninhibitor of IDO and an inhibitor of CTLA-4. In some embodiments, themethod comprises administering an inhibitor of IDO and an inhibitor ofthe PD-L1/PD-1 pathway. In some embodiments, the method comprisesadministering an inhibitor of IDO, an inhibitor of the PD-L1/PD-1pathway, and an inhibitor of CTLA-4. In some embodiments, the methodcomprises administering an inhibitor of the PD-L1/PD-1 pathway and aninhibitor of CTLA-4.

The IDO inhibitor may be any effective IDO inhibitor. In sonicembodiments, the inhibitor of IDO is a compound of Formula I:

or a pharmaceutically acceptable salt thereof, wherein R¹ is NH₂ or CH₃,R² is Cl, F, CF₃, CH₃, Br, or CN, R³ is H or F, R⁴ is H or CH₃, and, nis 1 or 2. In some embodiments, the inhibitor of IDO is the compound of:

The inhibitor of the PD-L1/PD-1 pathway may be any effective inhibitorof the PD-L1/PD-1 pathway. In some embodiments, the inhibitor of thePD-L1/PD-1 pathway is an anti-PD-L1 antibody or an anti-PD-1 antibody.In some embodiments, the anti-PD-L1 or anti-PD-1 antibody is amonoclonal antibody. In some embodiments, the monoclonal antibody is ahuman antibody. In some embodiments, the anti-PD-L1 or anti-PD-1antibody is a humanized antibody. In some embodiments, the anti-PD-L1monoclonal antibody is BMS-936559, MPDL3280A, BMS-936558, MK-3475,CT-011, or MEDI4736.

The inhibitor of CTLA-4 may be any effective inhibitor of CTLA-4. Insome embodiments, the inhibitor of CTLA-4 is an anti-CTLA-4 antibody. Insome embodiments, the anti-CTLA-4 antibody is a monoclonal antibody. Insome embodiments, the anti-CTLA-4 antibody monoclonal antibody is ahuman antibody. In some embodiments, the anti-CTLA-4 antibody is ahumanized antibody. In some embodiments, the anti-CTLA-4 monoclonalantibody is ipilimumab.

“Treatment” or “treating” includes (1) inhibiting a disease in a subjector patient experiencing or displaying the pathology or symptomatology ofthe disease (e.g., arresting further development of the pathology and/orsymptomatology), (2) ameliorating a disease in a subject or patient thatis experiencing or displaying the pathology or symptomatology of thedisease (e.g., reversing the pathology and/or symptomatology), and/or(3) effecting any measurable decrease in a disease in a subject orpatient that is experiencing or displaying the pathology orsymptomatology of the disease. In some embodiments, treating cancer isfurther defined as reducing the size of a tumor or inhibiting growth ofa tumor.

The compounds may be administered by any acceptable route. In someembodiments, the compounds are administered orally, intraadiposally,intraarterially, intraarticularly, intracranially, intradermally,intralesionally, intramuscularly, intranasally, intraocularally,intrapericardially, intraperitoneally, intrapleurally,intraprostaticaly, intrarectally, intrathecally, intratracheally,intratumorally, intraumbilically, intravaginally, intravenously,intravesicularlly, intravitreally, liposomally, locally, mucosally,orally, parenterally, rectally, subconjunctival, subcutaneously,sublingually, topically, transbuccally, transdermally, vaginally, incremes, in lipid compositions, via a catheter, via a lavage, viacontinuous infusion, via infusion, via inhalation, via injection, vialocal delivery, via localized perfusion, bathing target cells directly,or any combination thereof. In specific embodiments, the inhibitoripilimumab is administered intraveneously.

A dose may be administered on an as needed basis or every 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 18, or 24 hours (or any range derivable therein)or 1, 2, 3, 4, 5, 6, 7, 8, 9, or times per day (or any range derivabletherein). A dose may be first administered before or after signs of aninfection are exhibited or felt by a patient or after a clinicianevaluates the patient for an infection. In some embodiments, the patientis administered a first dose of a regimen 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12 hours (or any range derivable therein) or 1, 2, 3, 4, or 5 daysafter the patient experiences or exhibits signs or symptoms of aninfection (or any range derivable therein). The patient may be treatedfor 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days (or any range derivabletherein) or until symptoms of an infection have disappeared or beenreduced or after 6, 12, 18, or 24 hours or 1, 2, 3, 4, or 5 days aftersymptoms of an infection have disappeared or been reduced. In specificembodiments, the inhibitor ipilimumab is administered every three weeks.

The at least two inhibitors may be in a single composition or may be inseparate compositions. If the inhibitors are in separate compositions,they may be administered simultaneously or with a delay betweenadministrations. In some embodiments, second or subsequent inhibitorsmay be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40,50, or 60 minutes or longer (or any range derivable therein) after thefirst inhibitor is administered. In some embodiments, second orsubsequent inhibitors may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, or 24 hours or longer (or any range derivable therein) after thefirst inhibitor is administered. In some embodiments, subsequentinhibitors may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or30 days or longer (or any range derivable therein) after the firstinhibitor is administered. In some embodiments, subsequent inhibitorsmay be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40,or 50 weeks or longer (or any range derivable therein) after the firstinhibitor is administered. In some embodiments, subsequent inhibitorsmay be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years or longer (orany range derivable therein) after the first inhibitor is administered.In some embodiments, the first inhibitor is an inhibitor of IDO and thesecond inhibitor is an inhibitor of the PD-L1/PD-1 pathway. In someembodiments, the first inhibitor is an inhibitor of IDO and the secondinhibitor is an inhibitor of CTLA-4. In some embodiments, the firstinhibitor is an inhibitor of the PD-L1/PD-1 pathway and the secondinhibitor is an inhibitor of IDO. In some embodiments, the firstinhibitor is an inhibitor of the PD-L1/PD-1 pathway and the secondinhibitor is an inhibitor of CTLA-4. In some embodiments, the firstinhibitor is an inhibitor of CTLA-4 and the second inhibitor is aninhibitor of the PD-L1/PD-1 pathway. In some embodiments, the firstinhibitor is an inhibitor of CTLA-4 and the second inhibitor is aninhibitor of IDO.

The cancer may be any cancer. In some embodiments, the cancer ismelanoma, cervical cancer, breast cancer, ovarian cancer, prostatecancer, testicular cancer, urothelial carcinoma, bladder cancer,non-small cell lung cancer, small cell lung cancer, sarcoma, colorectaladenocarcinoma, gastrointestinal stromal tumors, gastroesophagealcarcinoma, colorectal cancer, pancreatic cancer, kidney cancer,hepatocellular cancer, malignant mesothelioma, leukemia, lymphoma,myelodysplastic syndrome, multiple myeloma, transitional cell carcinoma,neuroblastoma, plasma cell neoplasms, Wilm's tumor, glioblastoma,retinoblastoma, or hepatocellular carcinoma. In some embodiments, thecancer is melanoma.

The compositions may be administered one or more times. In someembodiments, the compositions are administered 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 times or more. In specific embodiments, the inhibitoripilimumab is administered 4 times.

Methods may be used in combination with additional cancer therapy. Insome embodiments, the distinct cancer therapy comprises surgery,radiotherapy, chemotherapy, toxin therapy, immunotherapy, cryotherapy orgene therapy. In some embodiments, the cancer is achemotherapy-resistant or radio-resistant cancer.

In some aspects, provided herein are methods of monitoring the patient'sresponse to the treatments disclosed herein. In particular embodiments,disclosed are methods for monitoring the treatment of cancer by any ofthe methods disclosed herein comprising determining the level of IL-2 ina first sample from the subject, determining the level of IL-2 in asecond sample from the subject, and comparing the level of IL-2 in thefirst sample to the level of IL-2 in the second sample, wherein a levelof IL-2 in the second sample that is greater than the level of IL-2 inthe first sample indicates that the patient is responding to thetreatment. The first sample may be obtained at any time before or aftertreatment begins. In some embodiments, the first sample is obtainedbefore treatment begins. In some embodiments, the first sample isobtained at the time treatment begins. In some embodiments, the firstsample is obtained after treatment is administered. The second samplemay be obtained at any time after treatment is administered. In someembodiments, the second sample is obtained 1, 2, 3, 4, 5, 6, 7, or moredays, 1, 2, 3, 4, or more weeks, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12or more months after treatment begins. In some embodiments, the secondsample is obtained three days after treatment is administered. In someembodiments, samples may be obtained from several tumor sites.

“Effective amount” or “therapeutically effective amount” or“pharmaceutically effective amount” means that amount which, whenadministered to a subject or patient for treating a disease, issufficient to effect such treatment for the disease. In someembodiments, the subject is administered at least about 0.01, 0.02,0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, 1.0, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70,80, 90, or 100 mg/kg (or any range derivable therein). In specificembodiments, 50 mg/10 mL (5 mg/mL) of the inhibitor ipilimumab isadministered. In specific embodiments, 200 mg/40 mL (5 mg/mL) of theinhibitor ipilimumab is administered.

In other aspects, embodiments provide compositions comprising at leasttwo of the following: an inhibitor of indoleamine-2,3-dioxygenase (IDO),an inhibitor of the PD-L1/PD-1 pathway, an inhibitor of CTLA-4, aninhibitor of CD25, or IL-7. In some embodiments, the compositioncomprises an inhibitor of IDO and an inhibitor of CTLA-4. In someembodiments, the composition comprises an inhibitor of IDO and aninhibitor of the PD-L1/PD-1 pathway. In some embodiments, thecomposition comprises an inhibitor of IDO, an inhibitor of thePD-L1/PD-1 pathway, and an inhibitor of CTLA-4. In some embodiments, thecomposition comprises an inhibitor of the PD-L1/PD-1 pathway and aninhibitor of CTLA-4. It is contemplated that compositions may bepharmaceutical compositions or may be pharmaceutically acceptablecompositions.

As used herein, “hydrogen” means —H; “hydroxy” means OH; “oxo” means ═O;“halo” means independently —F, —Cl, —Br or —I; “amino” means —NH₂ (seebelow for definitions of groups containing the term amino, e.g.,alkylamino); “hydroxyamino” means —NHOH; “nitro” means —NO₂; imino means═NH (see below for definitions of groups containing the term imino,e.g., alkylamino); “cyano” means —CN; “azido” means N3; “mercapto” means—SH; “thio” means ═S; “sulfonamido” means —NHS(O)₂— (see below fordefinitions of groups containing the term sulfonamido, e.g.,alkylsulfonamido); “sulfonyl” means —S(O)₂— (see below for definitionsof groups containing the term sulfonyl, e.g., alkylsulfonyl); and“silyl” means —SiH₃ (see below for definitions of group(s) containingthe term silyl, e.g., alkylsilyl).

For the groups below, the following parenthetical subscripts furtherdefine the groups as follows: “(Cn)” defines the exact number (n) ofcarbon atoms in the group; “(C≤n)” defines the maximum number (n) ofcarbon atoms that can be in the group; (Cn-n′) defines both the minimum(n) and maximum number (n′) of carbon atoms in the group. For example,“alkoxy_((C≤10))” designates those alkoxy groups having from 1 to 10carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or any rangederivable therein (e.g., 3-10 carbon atoms)). Similarly,“alkyl_((C2-10))” designates those alkyl groups having from 2 to 10carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, or any rangederivable therein (e.g., 3-10 carbon atoms)).

In addition, atoms making up the compounds of the present embodimentsare intended to include all isotopic forms of such atoms. Isotopes, asused herein, include those atoms having the same atomic number butdifferent mass numbers. By way of general example and withoutlimitation, isotopes of hydrogen include tritium and deuterium, andisotopes of carbon include ¹³C and ¹⁴C. Similarly, it is contemplatedthat one or more carbon atom(s) of a compound described herein may bereplaced by a silicon atom(s). Further, it is contemplated that anyoxygen atom discussed in any compound herein may be replaced by a sulfuror selenium atom.

Any undefined valency on an atom of a structure shown in thisapplication implicitly represents a hydrogen atom bonded to the atom.

The use of the word “a” or “an,” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

The terms “comprise,” “have” and “include” are open-ended linking verbs.Any forms or tenses of one or more of these verbs, such as “comprises,”“comprising,” “has,” “having,” “includes” and “including,” are alsoopen-ended. For example, any method that “comprises,” “has” or“includes” one or more steps is not limited to possessing only those oneor more steps and also covers other unlisted steps.

The term “effective,” as that term is used in the specification and/orclaims, means adequate to accomplish a desired, expected, or intendedresult.

The term “hydrate” when used as a modifier to a compound means that thecompound has less than one (e.g., hemihydrate), one (e.g., monohydrate),or more than one (e.g., dehydrate) water molecules associated with eachcompound molecule, such as in solid forms of the compound.

As used herein, the term “IC₅₀” refers to an inhibitory dose which is50% of the maximum response obtained.

An “isomer” of a first compound is a separate compound in which eachmolecule contains the same constituent atoms as the first compound, butwhere the configuration of those atoms in three dimensions differs.

As used herein, the term “patient” or “subject” refers to a livingmammalian organism, such as a human, monkey, cow, sheep, goat, dogs,cat, mouse, rat, guinea pig, or transgenic species thereof. In certainembodiments, the patient or subject is a primate. Non-limiting examplesof human subjects are adults, juveniles, infants and fetuses.

“Pharmaceutically acceptable” means that which is useful in preparing apharmaceutical composition that is generally safe, non-toxic and neitherbiologically nor otherwise undesirable and includes that which isacceptable for veterinary use as well as human pharmaceutical use.

“Pharmaceutically acceptable salts” means salts of compounds that arepharmaceutically acceptable, as defined above, and that possess thedesired pharmacological activity. Such salts include acid addition saltsformed with inorganic acids such as hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid, phosphoric acid, and the like; or withorganic acids such as 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonicacid, 2-naphthalenesulfonic acid, 3-phenylpropionic acid,4,4′-methylenebis(3-hydroxy-2-ene-1-carboxylic acid),4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, acetic acid,aliphatic mono- and dicarboxylicacids, aliphatic sulfuric acids,aromatic sulfuric acids, benzenesulfonic acid, benzoic acid,camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid,cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid,glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid,heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid,laurylsulfuric acid, maleic acid, malic acid, malonic acid, mandelicacid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoicacid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substitutedalkanoic acids, propionic acid, p-toluenesulfonic acid, pyruvic acid,salicylic acid, stearic acid, succinic acid, tartaric acid,tertiarybutylacetic acid, trimethylacetic acid, and the like.Pharmaceutically acceptable salts also include base addition salts whichmay be formed when acidic protons present are capable of reacting withinorganic or organic bases. Acceptable inorganic bases include sodiumhydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide andcalcium hydroxide. Acceptable organic bases include ethanolamine,diethanolamine, triethanolamine, tromethamine, N-methylglucamine and thelike. It should be recognized that the particular anion or cationforming a part of any salt of described embodiments is not critical, solong as the salt, as a whole, is pharmacologically acceptable.Additional examples of pharmaceutically acceptable salts and theirmethods of preparation and use are presented in Handbook ofPharmaceutical Salts: Properties, and Use (P. H. Stahl & C. G. Wermutheds., Verlag Helvetica Chimica Acta, 2002),

“Prevention” or “preventing” includes: (1) inhibiting the onset of adisease in a subject or patient which may be at risk and/or predisposedto the disease but does not yet experience or display any or all of thepathology or symptomatology of the disease, and/or (2) slowing the onsetof the pathology or symptomatology of a disease in a subject of patientwhich may be at risk and/or predisposed to the disease but does not yetexperience or display any or all of the pathology or symptomatology ofthe disease.

“Prodrug” means a compound that is convertible in vivo metabolicallyinto an inhibitor according to embodiments described herein. The prodrugitself may or may not also have activity with respect to a given targetprotein. For example, a compound comprising a hydroxy group may beadministered as an ester that is converted by hydrolysis in vivo to thehydroxy compound. Suitable esters that may be converted in vivo intohydroxy compounds include acetates, citrates, lactates, phosphates,tartrates, malonates, oxalates, salicylates, propionates, succinates,fumarates, maleates, methylene-bis-β-hydroxynaphthoate, gentisates,isethionates, di-p-toluoyltartrates, methanesulfonates,ethanesulfonates, benzenesulfonates, p-toluenesulfonates,cyclohexylsulfamates, quinates, esters of amino acids, and the like.Similarly, a compound comprising an amine group may be administered asan amide that is converted by hydrolysis in vivo to the amine compound.

A “stereoisomer” or “optical isomer” is an isomer of a given compound inwhich the same atoms are bonded to the same other atoms, but where theconfiguration of those atoms in three dimensions differs. “Enantiomers”are stereoisomers of a given compound that are mirror images of eachother, like left and right hands. “Diastereomers” are stereoisomers of agiven compound that are not enantiomers.

As used herein, the term “water soluble” means that the compounddissolves in water at least to the extent of 0.010 mole/liter or isclassified as soluble according to literature precedence.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1 illustrates the expression of FoxP3, PD-L1, and IDO in individualmelanoma metastases as assessed by qRT-PCR.

FIG. 2 illustrates the anti-CTLA-4 blockade, alone or in combinationwith other inhibitors. Measurement of tumor growth initiated 7 dayspost-tumor inoculation and continued throughout the experiment. Depictedare all time points until first animals had to be sacrificed. Two-wayANOVA test comparing each combination to anti-CTLA4 treatment.

FIGS. 3A-B illustrates the anti-PD-L1 blockade, alone or in combinationwith other inhibitors. (A) Tumor measurement over time. (B) Anti-CTLA-4regime and sequential administration of anti-PD-L1 starting either onday 4, day 10, or day 14 post-tumor inoculation. Time points aredisplayed until sacrifice of first animal. Two-way ANOVA test comparingeach combination to anti-PD-L1 treatment.

FIGS. 4A-B illustrates the effects of IDO inhibition. (A) Tumormeasurement over time. (B) Anti-CTLA-4 regime and sequentialadministration of IDOi starting either on day 4, day 10, or day 14post-tumor inoculation. Time points are displayed until sacrifice offirst animal. Two-way ANOVA test comparing each combination to thematching IDOi treatment.

FIGS. 5A-B illustrates the effects of CD25-depletion. (A) Tumoroutgrowth measured over time. (B) Combinations of anti-CTLA-4+anti-PD-L1or IDOi, in combination with anti-CD25-mediated Treg depletion(circles). Time points are displayed until sacrifice of first animal.Two-way ANOVA test comparing each combination to anti-CD25 treatment orcombination.

FIG. 6 illustrates the impact on T cell numbers. Flow cytometricanalysis was performed at day of sacrifice determined by either tumorsize or experimental endpoint at day 35 post-tumor injection.*significantly different to non-treated control (Mann Whitney U test).

FIG. 7 illustrates the impact on T cell function. IFN-γ ELISpot wasperformed using splenocytes from day 35 or earlier. 2×10⁶ splenocyteswere cultured for 24 h with or w/o STY peptide. Depicted are means(+/−SEM) of 4 mice. The red number indicates the number of spotsrelative to non-stimulated control and two representative examples aredisplayed.

FIG. 8 illustrates expression of LAG-3 and 4-1BB on anergic T cells asassessed by flow cytometry.

FIGS. 9A-C illustrate tumor outgrowth in response to single and pairwisecombinations of αCTLA-4, αPD-L1 and IDOi. Tumor outgrowth measured inmm² comparing single treatment to the respective combined doubletreatment of αCTLA-4 and αPD-L1 C (A), αCTLA-4 and IDOi (B), and αPD-L1and IDOi (C).

FIGS. 10A-C depicts the incidence of tumor-reactive, SIY-specific CD8⁺ Tcells in the tumor-draining lymph node (TdLN) and in the spleen.Peptide/K^(b) pentamer staining was performed on gated CD3+CD8+ T cells,isolated from TdLN (A) or spleen (B). (C) The functional capacity ofthese cells was further assessed by IFN-γ ELISpot.

FIGS. 11A-C illustrates double treatments restore capacity oflymphocytes within the tumor to produce IL-2 and proliferate. (A) Arepresentative FACS plot showing proliferation via cell trace dilutionon the x-axis and intracellular IL-2 staining on the y-axis. (B) Doubletreatments show a significant increase in proliferation compared tonon-stimulated and single treatments when tested with a one-way Anova.(C) The percentages of IFN-γ⁺ (open bar), IFN-γ⁺ and IL-2⁺ (gray bar),and proliferating IFN-γ⁺IL-2⁺ cells (filled bar) were calculated withinthe CD3⁺CD8⁺ cell population.

FIGS. 12A-B are the non-stimulated controls corresponding to FIGS. 11(A) and (C).

FIGS. 13A-C demonstrates restoration of IL-2 production andproliferation of tumor-infiltrating lymphocytes in the absence of new Tcell migration. (A) B16-SIY bearing mice were either treated with FTY720or control vehicle prior to initiation of therapy, to prevent migrationof new lymphocytes into the tumor. Peripheral blood T cell numbersfollowing FTY720 treatment on the day of tumor harvest for analysis.Open bars depict the number of CD45⁺CD3⁺ T cells detected in 200 ulperipheral blood of vehicle treated mice set to 100%. Filled barsrepresent the number found in FTY720-treated mice, relative to thevehicle-treated group. Single cell suspensions from tumor were labeledwith cell trace and stimulated with plate-bound anti-CD3 antibody for 48h prior then with anti-CD3 and anti-CD28 in the presence of Brefeldin A.Cells were then analyzed for proliferation by cell trace dilution andproduction of IL-2 via intracellular staining. Depicted are thepercentages of proliferating cells (B) or proliferating and IL-2producing cells (C) comparing vehicle-treated groups (open bar) toFTY720-treated groups (filled bar).

FIGS. 14A-B demonstrates the immunotherapy doublets that result inincreased BrdU incorporation by CD8+ and CD4+ tumor-infiltrating T cellsin vivo. Tumor-infiltrating lymphocytes were harvested on day 7, 24 hafter a single BrdU pulse in vivo, and cells were stained for BrdU alongwith anti-CD3, anti-CD4, and anti-CD8. Depicted are percentages of BrdU⁺cells that were CD3⁺ CD8⁺ (A) and CD3⁺ CD4⁺ (B).

FIGS. 15A-B illustrates in vivo proliferation of CD8+ and CD4+ positiveT cells in spleen (A) and TdLN (B).

FIG. 16 illustrates immunotherapy doublets that result in increasedfrequencies of tumor antigen-specific T cells at later time points inthe periphery. Depicted is an IFN-γ ELISpot of splenocytes harvested onday 14 with open bars being the un-stimulated control and filled barsrepresenting SIY-stimulation.

FIG. 17 demonstrates immunotherapy doublets increase frequency andpersistence of SIY/K^(b) pentamer-specific T cells in the periphery andin the tumor.

FIGS. 18A-B illustrates IDOi tumor outgrowth in response to singleαCTLA-4, αPD-L1, IDOc and IDOi treatment (A) and pairwise (B)combinations of αCTLA-4 and αPD-L1 with IDOc and IDOi. Results wereanalyzed using a 2 way Anova comparing the single to double regimes aswell as the double regimes to each other.

DETAILED DESCRIPTION

In some embodiments, the methods described herein involve the use of acombination of at least two of the following: an inhibitor ofindoleamine-2,3-dioxygenase (IDO), an inhibitor of the PD-L1/PD-1pathway, an inhibitor of CTLA-4, an inhibitor of CD25, or IL-7. Theinventors particularly observed a major synergistic effect of combininganti-CTLA-4 with either an IDO inhibitor, with anti-PD-L1 mAb, or withCD-25 depletion. Such combinations have been found to demonstrate asynergistic effect in treating cancer and tumors, for example byreducing tumor size, increasing the percentage of antigen-specific Tcells, and increasing T cell function.

A. Synergistic Therapeutic Effects

Human Melanoma Metastases Co-Express PD-L1, IDO, and Tregs, and ShowSigns of Classical Anergy.

The inventors performed a detailed analysis of the tumormicroenvironment utilizing biopsies from patients with metastaticmelanoma and identified two major subsets of tumors. One subset has abroad signature of inflammation that includes T cell markers andchemokines for lymphocyte recruitment, while the second subset is“bland” and lacks this inflammatory profile (Harlin et al., 2009).Clinical responses to melanoma vaccines appear to fall within the firstsubset, implying that non-inflamed tumors that lack chemokines might notbe capable of supporting recruitment of activated T cells to enabletumor rejection (Gajewski et al., 2010). However, the question arises asto why the inflamed tumor phenotype that includes activated CD8+ T cellsdoes not get rejected spontaneously by the host immune system. In fact,the inventors found that these tumors also show evidence of at least 4immune inhibitory mechanisms: expression of PD-L1, presence of FoxP3+Tregs, T cell anergy, and expression of IDO (Gajewski et al., 2006). Apositive correlation was observed between the extent of the T cellinfiltrate and the expression of PD-L1, FoxP3, and IDO (FIG. 1). Ananergic phenotype was implied by lack of expression of B7-1 and B7-2within the tumor microenvironment, and evidence for antigen-specificintrinsic T cell dysfunction (Harlin et al., 2006). Together, theseresults argue that immune suppressive mechanisms may dominate in thetumor microenvironment and prevent immune-mediated tumor elimination.

Treg Depletion and Reversal of Anergy Through Homeostatic ProliferationAct Synergistically to Control B16 Melanoma In Vivo.

The inventors have found that blockade of the PD-L1/PD-1 pathway, eitherusing PD-1 knockout T cells or an anti-PD-L1 mAb, can have a positiveimpact on T cell-mediated tumor control (Blank et al., 2004; Zhang etal., 2009). Similarly, depletion of Tregs alone can have some impact(Kline et al., 2008), as can reversal of T cell anergy throughhomeostatic proliferation (Brown et al., 2006). However, in the B16melanoma model, these effects are usually partial and don't result incomplete tumor elimination. Interestingly, when Treg depletion wascombined with homeostatic proliferation, complete eradication of B16melanoma was observed (Kline et al., 2008). These results indicate thatthe combinatorial blockade of two or more regulatory pathways have asynergistic effect, thus enabling improved tumor control. Inexperiments, the inventors also have observed that combined blockade ofPD-1/PD-L1 interactions plus homeostatic proliferation can also besynergistic.

Anergic T Cells Express LAG3, Tim3, and 4-1BB.

Inasmuch as classical T cell anergy appears to be operational as onemechanism of immune escape in the tumor context, a detailed analysis ofthe molecular mechanisms controlling the anergic, dysfunctional statewas performed. The inventors determined that the transcriptionalregulator Egr2 drives expression of the anergy program, which includesgenes encoding inhibitory signaling molecules such as DGK-α (Zha et al.,2006) and Cb1-b (unpublished data). The inventors utilized conditionalEgr2-KO mice to demonstrate that T cells consequently become anergyresistant and show improved tumor control in vivo. Through an analysisof the entire Egr2-driven transcriptome in anergy, the inventorsidentified new surface markers that may help characterize the anergicphenotype but also may allow manipulation of anergic cells to restoretheir function. Two of these markers are LAG-3 and 4-1BB (FIG. 8). Moredetailed phenotyping has also revealed expression of Tim-3. Both LAG-3and Tim3 are inhibitory molecules that can contribute to the blunting ofT cell activation. Data indicates 4-1BB is a costimulatory receptor thatmight allow restoration of the function of anergic cells once ligated.Analysis of CD8+ T cells infiltrating B16 melanoma has revealedco-expression of all of these surface markers, on a subset of cellsexpression PD-1. Therefore, the administration of mAbs against LAG-3,4-1BB, and Tim3 can also be performed in concert with IDO inhibition.

B. Indoleamine-2,3-Dioxygenase (IDO)

Tryptophan (Trp) is an essential amino acid required for thebiosynthesis of proteins, niacin and the neurotransmitter5-hydroxytryptamine (serotonin). IDO catalyzes the first and ratelimiting step in the degradation of L-tryptophan to N-formyl-kynurenine.In human cells, a depletion of Trp resulting from IDO activity is aprominent gamma interferon (IFN-γ)-inducible antimicrobial effectormechanism. IFN-γ stimulation induces activation of IDO, which leads to adepletion of Trp, thereby arresting the growth of Trp-dependentintracellular pathogens such as Toxoplasma gondii and Chlamydiatrachomatis. IDO activity also has an antiproliferative effect on manytumor cells, and IDO induction has been observed in vivo duringrejection of allogeneic tumors, indicating a possible role for thisenzyme in the tumor rejection process (Daubener 1999; Taylor 1991).

It has been observed that HeLa cells co-cultured with peripheral bloodlymphocytes (PBLs) acquire an immuno-inhibitory phenotype throughup-regulation of IDO activity. A reduction in PBL proliferation upontreatment with interleukin-2 (IL2) was believed to result from IDOreleased by the tumor cells in response to IFN-γ secretion by the PBLs.This effect was reversed by treatment with 1-methyl-tryptophan (1MT), aspecific IDO inhibitor. It was proposed that IDO activity in tumor cellsmay serve to impair antitumor responses (Logan 2002).

Further evidence for a tumoral immune resistance mechanism based ontryptophan degradation by IDO comes from the observation that most humantumors constitutively express IDO, and that expression of IDO byimmunogenic mouse tumor cells prevents their rejection by preimmunizedmice. This effect is accompanied by a lack of accumulation of specific Tcells at the tumor site and can be partly reverted by systemic treatmentof mice with an inhibitor of IDO, in the absence of noticeable toxicity.Thus, it was suggested that the efficacy of therapeutic vaccination ofcancer patients might be improved by concomitant administration of anIDO inhibitor (Uyttenhove et al., 2003). It has also been shown that theIDO inhibitor, 1-MT, can synergize with chemotherapeutic agents toreduce tumor growth in mice, suggesting that IDO inhibition may alsoenhance the anti-tumor activity of conventional cytotoxic therapies(Muller et al., 2005).

One mechanism contributing to immunologic unresponsiveness toward tumorsmay be presentation of tumor antigens by tolerogenic host APCs. A subsetof human DO-expressing antigen-presenting cells (APCs) that coexpressedCD 123 (IL3RA) and CCR6 and inhibited T-cell proliferation have alsobeen described. Both mature and immature CD123-positive dendritic cellssuppressed T-cell activity, and this IDO suppressive activity wasblocked by 1MT (Munn et al., 2002). It has also been demonstrated thatmouse tumor-draining lymph nodes (TDLNs) contain a subset ofplasmacytoid dendritic cells (pDCs) that constitutively expressimmunosuppressive levels of IDO. Despite comprising only 0.5% of lymphnode cells, in vitro, these pDCs potently suppressed T cell responses toantigens presented by the pDCs themselves and also, in a dominantfashion, suppressed T cell responses to third-party antigens presentedby nonsuppressive APCs. Within the population of pDCs, the majority ofthe functional IDO-mediated suppressor activity segregated with a novelsubset of pDCs coexpressing the B-lineage marker CD19. Thus, it washypothesized that IDO-mediated suppression by pDCs in TDLNs creates alocal microenvironment that is potently suppressive of host antitumor Tcell responses (Munn et al., 2004).

IDO degrades the indole moiety of tryptophan, serotonin and melatonin,and initiates the production of neuroactive and immunoregulatorymetabolites, collectively known as kynurenines. By locally depletingtryptophan and increasing proapoptotic kynurenines, IDO expressed bydendritic cells (DCs) can greatly affect T-cell proliferation andsurvival. IDO induction in DCs could be a common mechanism of deletionaltolerance driven by regulatory T cells. Because such tolerogenicresponses can be expected to operate in a variety of physiopathologicalconditions, tryptophan metabolism and kynurenine production mightrepresent a crucial interface between the immune and nervous systems(Grohmann, et al., 2003, Trends Immunol., 24: 242-8). In states ofpersistent immune activation, availability of free serum Trp isdiminished and, as a consequence of reduced serotonin production,serotonergic functions may also be affected (Wirleitner et al., 2003).

Small molecule inhibitors of IDO are being developed to treat or preventIDO-related diseases. For example, oxadiazole and other heterocyclic IDOinhibitors are reported in US 2006/0258719 and US 2007/0185165. PCTPublication WO 99/29310 reports methods for altering T cell-mediatedimmunity comprising altering local extracellular concentrations oftryptophan and tryptophan metabolites, using an inhibitor of IDO such as1-methyl-DL-tryptophan, p-(3-benzofuranyl)-DL-alanine,p-[3-benzo(b)thienyl]-DL-alanine, and 6-nitro-L-tryptophan) (Munn etal., 1999). Reported in WO 03/087347, also published as European Patent1501918, are methods of making antigen-presenting cells for enhancing orreducing T cell tolerance (Munn et al., 2003). Compounds havingindoleamine-2,3-dioxygenase (IDO) inhibitory activity are furtherreported in WO 2004/094409; and U.S. Patent Application Publication No.2004/0234623 is directed to methods of treating a subject with a canceror an infection by the administration of an inhibitor ofindoleamine-2,3-dioxygenase in combination with other therapeuticmodalities. In some embodiments, the small molecule inhibitors are thosefound in the Examples below or disclosed in U.S. Pat. No. 8,088,803,which is incorporated in its entirety herein.

C. CTLA-4

CTLA4 (Cytotoxic T-Lymphocyte Antigen 4) is a CD28-family receptorexpressed on activated CD8+ and CD4+ T cells. It binds the same ligandsas CD28 (CD80 and CD86 on B cells and dendritic cells), but with higheraffinity than CD28. In contrast to CD28, which enhances cell functionwhen bound at the same time as the T cell receptor, CTLA4 inhibits Tcell functioning. CTLA4 blockade releases inhibitory controls on T cellactivation and proliferation, inducing antitumor immunity in bothpreclinical and early clinical trials (Quezada et al., 2006). The CTLA4pathway is the subject of much interest (see, for example, U.S. Pat. No.7,229,628).

Inhibitors of the CTLA4 pathway include, but are not limited toantibodies, peptides, nucleic acid molecules (including, for example, anantisense molecule, a PNA, or an RNAi), peptidomimetics, smallmolecules, a soluble CTLA4 ligand polypeptide, or a chimeric polypeptide(for example, a chimeric CTLA4 ligand/immunoglobulin molecule). Anantibody may be an intact antibody, an antibody binding fragment, or achimeric antibody. A chimeric antibody may include both human andnon-human portions. An antibody may be a polyclonal or a monoclonalantibody. An antibody may be derived from a wide variety of species,including, but not limited to mouse and human. An antibody may be ahumanized antibody. An antibody may be linked to another functionalmolecule, for example, another peptide or protein, a toxin, aradioisotype, a cytotoxic agent, cytostatic agent, a polymer, such as,for example, polyethylene glycol, polypropylene glycol orpolyoxyalkenes. In some embodiments, a mixture or cocktail of variousinhibitors of the CTLA4 pathway may be administered.

Any of a variety of antibodies may be used, including, but not limitedto, any of those described herein and those commercially available, forexample, ipilimumab from Bristol-Myers Squibb and tremelimumab fromMedImmune. Other anti-CTLA4 antibodies include, but are not limited to,those taught in U.S. Pat. Nos. 7,311,910; 7,307,064; 7,132,281;7,109,003; 7,034,121; 6,984,720; and 6,682,736. In some embodiments, oneor more anti-CTLA4 antibodies may be humanized.

D. PD-L1/PD-1 Axis

Programmed death ligand 1 (PD-L1) is a 40 kDa type 1 transmembraneprotein that has been speculated to play a major role in suppressing theimmune system during particular events such as pregnancy, tissueallografts, autoimmune disease and other disease states such ashepatitis. Normally the immune system reacts to foreign antigens wherethere is some accumulation in the lymph nodes or spleen which triggers aproliferation of antigen-specific CD8+ T cell. The formation of PD-1receptor/PD-L1 ligand complex transmits an inhibitory signal whichreduces the proliferation of these CD8+ T cells at the lymph nodes andsupplementary to that PD-1 is also able to control the accumulation offoreign antigen specific T cells in the lymph nodes through apoptosiswhich is further mediated by a lower regulation of the gene Bcl-2. PD-L1binds to its receptor, PD-1, found on activated T cells, B cells, andmyeloid cells, to modulate activation or inhibition. The affinitybetween PD-L1 and PD-1, as defined by the dissociation constant Kd, is770 nM.

Inhibitors of the PD-L1/PD-1 pathway include, but are not limited to,antibodies, peptides, nucleic acid molecules (including, for example, anantisense molecule, a PNA, or an RNAi), peptidomimetics, smallmolecules, a soluble PD-1 ligand polypeptide, or a chimeric polypeptide(for example, a chimeric PD-1 ligand/Immunoglobulin molecule). Anantibody may be an intact antibody, an antibody binding fragment, or achimeric antibody. A chimeric antibody may include both human andnon-human portions. An antibody may be a polyclonal or a moncoclonalantibody. An antibody may be a derived from a wide variety of species,including, but not limited to mouse and human. An antibody may be ahumanized antibody. An antibody may be linked to another functionalmolecule, for example, another peptide or protein, a toxin, aradioisotype, a cytotoxic agent, cytostatic agent, a polymer, such as,for example, polyethylene glycol, polypropylene glycol orpolyoxyalkenes.

Any of a variety of antibodies may be used, including, but not limitedto, any of those described herein and those commercially available, forexample, BMS-936559 or BMS-936558 from Bristol-Myers Squibb, MPDL3280Afrom Genentech, MK-3475 from Merck, CT-011 from Curetech, and MEDI4736from MedImmune.

In some embodiments, one or more anti-PDL-1 antibodies may be humanized.

E. Antibodies

The term “antibody” as used herein refers to an immunoglobulin whichpossesses the ability to combine with an antigen. It comprises at leasttwo heavy (H) chains and two light (L) chains inter-connected bydisulfide bonds. Non-limiting examples of antibodies include monoclonalantibodies (e.g., full length or intact monoclonal antibodies),polyclonal antibodies, multivalent antibodies, and multi-specificantibodies (e.g., bi-specific antibodies as long as they exhibit thedesired biological activity). An antibody can be human, humanized oraffinity-matured, or combinations thereof.

The term “antibody fragment” comprises only a portion of an intactantibody, wherein the portion preferably retains at least one,preferably most or all, of the functions normally associated with thatportion when present in an intact antibody. Examples of antibodyfragments include Fab, Fab′, F(ab′)2, and Fv fragments; diabodies;linear antibodies; single-chain antibody molecules; and multispecificantibodies formed from antibody fragments. In one embodiment, anantibody fragment comprises an antigen binding site of the intactantibody and thus retains the ability to bind antigen. In anotherembodiment, an antibody fragment, for example one that comprises the Fcregion, retains at least one of the biological functions normallyassociated with the Fc region when present in an intact antibody. In oneembodiment, an antibody fragment is a monovalent antibody that has an invivo half-life substantially similar to an intact antibody. For example,such an antibody fragment may comprise an antigen-binding arm linked toan Fc sequence capable of conferring in vivo stability to the fragment.

An “isolated” antibody is one which has been identified and separated orrecovered, or both, from a component of its natural environment.Contaminant components of an isolated antibody's natural environment arematerials which would interfere with diagnostic or therapeutic uses ofthe antibody. Non-limiting examples of such contaminants includeenzymes, hormones, and other proteinaceous or non-proteinaceous solutes.In some embodiments, for example, the antibody may be purified togreater than 95% by weight of antibody as determined by the Lowrymethod, and sometimes more than 99% by weight. Isolated antibodyincludes the antibody in situ within recombinant cells because at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope. Themonoclonal antibodies herein specifically include “chimeric” antibodiesin which a portion of the heavy or light chain, or both, is identicalwith or homologous to corresponding sequences in antibodies derived froma particular species or belonging to a particular antibody class orsubclass, while the remainder of the chain or chains are identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies so long as they exhibit the desiredbiological activity.

The term “variable” refers to the fact that certain portions of thevariable domain sequences differ extensively among antibodies and areimportant to the binding and specificity of each particular antibody forits particular antigen. However, such variability is not evenlydistributed throughout the variable domains of antibodies and isconcentrated in three segments called complementarity-determiningregions (CDRs) or hypervariable regions which occur in both of thelight-chain and the heavy-chain variable domains. The more highlyconserved portions of variable domains are called the framework (FR).The variable domains of native heavy and light chains each comprise fourFR regions (largely adopting a beta-sheet configuration) connected bythree CDRs (which form loops connecting, and in some cases forming partof, the beta sheet structure). The CDRs in each chain are held togetherin close proximity by the FR regions and, with the CDRs from the otherchain, contribute to the formation of the antigen-binding site ofantibodies. The constant domains are not involved directly in binding anantibody to an antigen, but exhibit various effector functions, such asparticipation of the antibody in antibody-dependent cellular toxicity.

The term “hypervariable region,” “HVR,” or “HV,” when used herein,refers to the regions of an antibody variable domain which arehypervariable in sequence or form structurally defined loops, or both.Generally, antibodies comprise six hypervariable regions; three in theVH (H1, H2, H3), and three in the VL (L1, L2, L3).

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or non-human primate having the desired specificity,affinity, and capacity. In some instances, FR residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies may comprise residues that are notfound in the recipient antibody or in the donor antibody. Thesemodifications are made to further refine antibody performance. Ingeneral, the humanized antibody may comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the hypervariable loops correspond to those of anon-human immunoglobulin and all or substantially all of the FRs arethose of a human immunoglobulin sequence. The humanized antibodyoptionally may also comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin.

The term “recombinant human antibody,” as used herein, includes allhuman antibodies that are prepared, expressed, created or isolated byrecombinant means, such as (a) antibodies isolated from an animal (e.g.,a mouse) that is transgenic or trans-chromosomal for humanimmunoglobulin genes or a hybridoma prepared therefrom (describedfurther below), (b) antibodies isolated from a host cell transformed toexpress the human antibody, e.g., from a transfectoma, (c) antibodiesisolated from a recombinant, combinatorial human antibody library, and(d) antibodies prepared, expressed, created or isolated by any othermeans that involve splicing of human immunoglobulin gene sequences toother DNA sequences. Such recombinant human antibodies have variableregions in which the framework and CDR regions are derived from humangermline immunoglobulin sequences. In certain embodiments, however, suchrecombinant human antibodies can be subjected to in vitro mutagenesis(or, when an animal transgenic for human Ig sequences is used, in vivosomatic mutagenesis) and thus the amino acid sequences of the V_(H) andV_(L) regions of the recombinant antibodies are sequences that, whilederived from and related to human germline V_(H) and V_(L) sequences,may not naturally exist within the human antibody germline repertoire invivo. In other certain embodiments, pre-assembled trinucleotides areused in the chemical synthesis of the CDR sequences for such recombinanthuman antibodies.

“Chimeric” antibodies (immunoglobulins) have a portion of the heavy orlight chain, or both, that is identical or homologous to correspondingsequences in antibodies derived from a particular species or belongingto a particular antibody class or subclass, while the remainder of thechain or chains are identical or homologous to corresponding sequencesin antibodies derived from another species or belonging to anotherantibody class or subclass, as well as fragments of such antibodies, solong as they exhibit the desired biological activity. Humanized antibodyas used herein is a subset of chimeric antibodies.

“Single-chain Fv” or “scFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. Generally, the scFv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains which enables thescFv to form the desired structure for antigen binding.

An “antigen” is a predetermined antigen to which an antibody canselectively bind. The target antigen may be polypeptide, carbohydrate,nucleic acid, lipid, hapten or other naturally occurring or syntheticcompound.

An “epitope” is the portion of the antigen to which the antibodyselectively binds. For a polypeptide antigen, the epitope is generally apeptide portion of about four to ten amino acids.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, such fragments comprise a heavy-chain variabledomain (V_(H)) connected to a light-chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)-V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or has been madeusing any of the techniques for making human antibodies as disclosedherein, or both. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen-bindingresidues.

An “affinity-matured” antibody is one with one or more alterations inone or more CDRs thereof which result in an improvement in the affinityof the antibody for antigen, compared to a parent antibody which doesnot possess those alteration(s). For example, affinity-maturedantibodies may have nanomolar or even picomolar affinities for thetarget antigen. Affinity-matured antibodies are produced by procedureswell-known in the art.

A “blocking” antibody or an “antagonist” antibody is one which inhibitsor reduces biological activity of the antigen it binds. Preferredblocking antibodies or antagonist antibodies substantially or completelyinhibit the biological activity of the antigen.

1. General Methods for the Production of Antibodies and Nucleic AcidsEncoding Antibodies

Embodiments include isolated antibodies or antibody fragments that bindimmunologically to native cell surface-expressed CTLA4, PD-L1, or PD-1and isolated polynucleotides comprising sequences encoding one or moreantibodies or antibody fragments that bind to CTLA4, PD-L1, or PD-1.Embodiments also include pharmaceutical compositions that includeantibodies or antibody fragments that binds immunologically to nativecell surface-expressed CTLA4, PD-L1, or PD-1 and polynucleotidescomprising sequences encoding one or more antibodies or antibodyfragments that bind to CTLA4, PD-L1, or PD-1.

In particular embodiments the anti-CTLA4, anti-PD-1, or anti-PD-L1antibodies are monoclonal antibodies. Monoclonal antibodies can beproduced by a variety of techniques, such as by conventional monoclonalantibody methodology using standard somatic cell hybridizationtechniques and viral or oncogenic transformation of B lymphocytes.

Monoclonal antibodies may be obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally occurringmutations that may be present in minor amounts. Thus, the modifier“monoclonal” indicates the character of the antibody as not being amixture of discrete antibodies.

The anti-CTLA4, anti-PD-1, or anti-PD-L1 monoclonal antibodies can bemade using a hybridoma method, or may be made by recombinant DNA methodswell-known to those of ordinary skill in the art.

Regarding the hybridoma method, the first step is immunization of anappropriate host, as may be achieved by coupling a peptide orpolypeptide immunogen to a carrier. Exemplary carriers are keyholelimpet hemocyanin (KLH), bovine serum albumin (BSA, ovalbumin, mouseserum albumin and rabbit serum albumin). As also is well known in theart, the immunogenicity of a particular immunogen composition can beenhanced by the use of non-specific stimulators of the immune response,known as adjuvants.

The amount of immunogen composition used in the production of polyclonalantibodies varies upon the nature of the immunogen as well as the animalused for immunization. A variety of routes can be used to administer theimmunogen (subcutaneous, intramuscular, intradermal, intravenous andintraperitoneal). The production of polyclonal antibodies may bemonitored by sampling blood of the immunized animal at various pointsfollowing immunization. One or more additional booster injections may begiven. The process of boosting and titering is repeated until a suitabletiter is achieved. When a desired level of immunogenicity is obtained,the immunized animal can be bled and the serum isolated and stored,and/or the animal can be used to generate monoclonal antibodies.

Following immunization, somatic cells with the potential for producingantibodies, specifically B lymphocytes (B cells), are selected for usein the monoclonal antibody-generating protocol. These cells may beobtained from biopsied spleens or lymph nodes, or from circulatingblood. The antibody-producing B lymphocytes from the immunized animalare then fused with cells of an immortal myeloma cell, generally one ofthe same species as the animal that was immunized. Any one of a numberof myeloma cells may be used, as are known to those of skill in the art.For example, where the immunized animal is a mouse, one may useP3-X63/Ag8, X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U, MPC-11,MPC11-X45-GTG 1.7 and S194/5XX0 Bul; for rats, one may use R210.RCY3,Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 andUC729-6 are all useful in connection with human cell fusions. Oneparticular murine myeloma cell is the NS-1 myeloma cell line (alsotermed P3-NS-1-Ag4-1), which is readily available from the NIGMS HumanGenetic Mutant Cell Repository by requesting cell line repository numberGM3573. Another mouse myeloma cell line that may be used is the8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cellline. Additional fusion partner lines for use with human B cells includeKR12 (ATCC CRL-8658; K6H6/B5 (ATCC CRL-1823 SHM-D33 (ATCC CRL-1668) andHMMA2.5 (Posner et al., 1987). In a particular embodiment, the line usedto generate the antibody in this invertion is OUR-1.

Methods for generating hybrids of antibody-producing spleen or lymphnode cells and myeloma cells usually comprise mixing somatic cells withmyeloma cells in the presence of an agent or agents (chemical orelectrical) that promote the fusion of cell membranes.

The viable, fused hybrids may be differentiated from the parental,infused cells (particularly the infused myeloma cells that wouldnormally continue to divide indefinitely) by culturing in a selectivemedium. The selective medium is generally one that contains an agentthat blocks the de novo synthesis of nucleotides in the tissue culturemedia. Exemplary agents are aminopterin, methotrexate, and azaserine.Aminopterin and methotrexate block de novo synthesis of both purines andpyrimidines, whereas azaserine blocks only purine synthesis. Whereaminopterin or methotrexate is used, the media is supplemented withhypoxanthine and thymidine as a source of nucleotides (HAT medium).Where azaserine is used, the media is supplemented with hypoxanthine.

Hypoxanthine aminopterm thymidine (HAT) may be used as a selectionmedium. Only cells capable of operating nucleotide salvage pathways areable to survive in HAT medium. The myeloma cells are defective in keyenzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyltransferase (HPRT), and they cannot survive. The B cells can operatethis pathway, but they have a limited life span in culture and generallydie within about two weeks. Therefore, the only cells that can survivein the selective media are those hybrids formed from myeloma and Bcells.

Culturing provides a population of hybridomas from which specifichybridomas are selected. Typically, selection of hybridomas is performedby culturing the cells by single-clone dilution in microtiter plates,followed by testing the individual clonal supernatants (after about twoto three weeks) for the desired reactivity. The assay should besensitive, simple and rapid, such as radioimmunoassays, enzymeimmunoassays, cytotoxicity assays, plaque assays, dot immunobindingassays, and the like.

The selected hybridomas are then serially diluted and cloned intoindividual antibody-producing cell lines, which clones can then bepropagated indefinitely to provide monoclonal antibodies. The cell linesmay be exploited for monoclonal antibody production using any methodknown to those of ordinary skill in the art. In one example, a sample ofthe hybridoma can be injected (often into the peritoneal cavity) into ananimal (e.g., a mouse). Optionally, the animals are primed with ahydrocarbon, especially oils such as pristane (tetramethylpentadecane)prior to injection. When human hybridomas are used in this way, it isoptimal to inject immunocompromised mice, such as severe combinedimmunodeficient (SCID) mice, to prevent tumor rejection. The injectedanimal develops tumors secreting the specific monoclonal antibodyproduced by the fused cell hybrid. The body fluids of the animal, suchas serum or ascites fluid, can then be tapped to provide monoclonalantibodies in high concentration. The individual cell lines could alsobe cultured in vitro, where the monoclonal antibodies are naturallysecreted into the culture medium from which they can be readily obtainedin high concentrations.

In the hybridoma method, a mouse or other appropriate host animal isimmunized to elicit lymphocytes that produce or are capable of producingantibodies that will specifically bind to the protein used forimmunization. Antibodies to CTLA4, PD-L1, or PD-1 may generally beraised in animals by multiple subcutaneous (sc) or intraperitoneal (ip)injections of CTLA4, PD-L1, or PD-1 and an adjuvant. CTLA4, PD-L1, orPD-1 may be prepared using methods well-known in the art.

In some embodiments, lymphocytes may be immunized in vitro. Lymphocytesthen are fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell.

The hybridoma cells thus prepared may be seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against CTLA4, PD-L1, orPD-1. The binding specificity of monoclonal antibodies produced byhybridoma cells may be determined be techniques well-known to those inthe art, such as by immunoprecipitation or by an in vitro binding assay(e.g., radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay(ELISA)). The binding affinity of the monoclonal antibody can, forexample, be determined by a Scatchard analysis. After hybridoma cellsare identified that produce antibodies of the desired specificity,affinity, and/or activity, the clones may be subcloned by limitingdilution procedures and grown by standard methods. The monoclonalantibodies secreted by the subclones may be suitably separated from theculture medium by conventional immunoglobulin purification proceduressuch as, for example, protein A-Sepharose, hydroxylapatitechromatography, gel electrophoresis, dialysis, or affinitychromatography.

The anti-CTLA4, anti-PD-1, or anti-PD-L1 antibodies can be made by usingcombinatorial libraries, such as a phage display library, to screen forsynthetic antibody clones with the desired activity or activities. Inprinciple, synthetic antibody clones are selected by screening phagelibraries containing phage that display various fragments of antibodyvariable region (Fv) fused to phage coat protein. Such phage librariesare panned by affinity chromatography against the desired antigen.Clones expressing Fv fragments capable of binding to the desired antigenare adsorbed to the antigen and thus separated from the non-bindingclones in the library. In a certain embodiment, the anti-CTLA4,anti-PD-1, or anti-PD-L1 antibodies are produced in bacteria and thelibrary is screened using phage display to identify the antibody with ahigh affinity to CTLA4, PD-L1, or PD-1.

Monoclonal antibodies produced by any means may be further purified, ifdesired, using any technique known to those of ordinary skill in theart, such as filtration, centrifugation and various chromatographicmethods such as FPLC or affinity chromatography or any other methodknown to those of ordinary skill in the art.

Nucleic acids encoding antibody gene fragments may be obtained fromimmune cells harvested from humans or animals. If a library biased infavor of anti-CTLA4, anti-PD-1, or anti-PD-L1 clones is desired, thesubject is immunized with CTLA4, PD-L1, or PD-1 to generate an antibodyresponse, and spleen cells and/or circulating B cells or otherperipheral blood lymphocytes (PBLs) are recovered for libraryconstruction. Additional enrichment for anti-CTLA4, anti-PD-L1, oranti-PD-1 reactive cell populations can be obtained by using a suitablescreening procedure to isolate B cells expressing CTLA4, PD-L1, orPD-1-specific membrane bound antibody. Alternatively, the use of spleencells and/or B cells or other PBLs from an unimmunized donor provides abetter representation of the possible antibody repertoire, and alsopermits the construction of an antibody library using any animal (humanor non-human) species in which CTLA4, PD-L1, or PD-1 is not antigenic.For libraries incorporating in vitro antibody gene construction, stemcells are harvested from the subject to provide nucleic acids encodingunrearranged antibody gene segments. The immune cells of interest can beobtained from a variety of animal species, such as human, mouse, rat,etc. Nucleic acid encoding antibody variable gene segments are recoveredfrom the cells of interest and amplified.

Nucleic acid sequence encoding a CTLA4, PD-L1, or PD-1 polypeptide canbe designed using the amino acid sequence of the desired region ofCTLA4, PD-L1, or PD-1. Alternatively, the cDNA sequence (or fragmentsthereof) may be used. DNAs encoding CTLA4, PD-L1, or PD-1 can beprepared by a variety of methods known in the art. Followingconstruction of the DNA molecule encoding CTLA4, PD-L1, or PD-1, the DNAmolecule is operably linked to an expression control sequence in anexpression vector, such as a plasmid, wherein the control sequence isrecognized by a host cell transformed with the vector. Suitable vectorsfor expression in prokaryotic and eukaryotic host cells are known in theart. Optionally, the DNA encoding CTLA4, PD-L1, or PD-1 is operablylinked to a secretory leader sequence resulting in secretion of theexpression product by the host cell into the culture medium. Host cellsare transfected and preferably transformed with the expression orcloning vectors and cultured in conventional nutrient media modified asappropriate for inducing promoters, selecting transformants, oramplifying the genes encoding the desired sequences.

The purified CTLA4, PD-L1, or PD-1 can be attached to a suitable matrixsuch as agarose beads, acrylamide beads, glass beads, cellulose, variousacrylic copolymers, hydroxyl methacrylate gels, polyacrylic andpolymethacrylic copolymers, nylon, neutral and ionic carriers, and thelike, for use in the affinity chromatographic separation of phagedisplay clones. Alternatively, CTLA4, PD-L1, or PD-1 can be used to coatthe wells of adsorption plates, expressed on host cells affixed toadsorption plates or used in cell sorting, or conjugated to biotin forcapture with streptavidin-coated beads, or used in any other art-knownmethod for panning phage display libraries. The phage library samplesare contacted with immobilized CTLA4, PD-L1, or PD-1 under conditionssuitable for binding of at least a portion of the phage particles withthe adsorbent. Normally, the conditions, including pH, ionic strength,temperature and the like are selected to mimic physiological conditions.The phages bound to the solid phase are washed and then eluted.Moreover, the enriched phages can be grown in bacterial culture andsubjected to further rounds of selection.

DNA encoding the hybridoma-derived monoclonal antibodies or phagedisplay Fv clones is readily isolated and sequenced using conventionalprocedures (e.g. by using oligonucleotide primers designed tospecifically amplify the heavy and light chain coding regions ofinterest from hybridoma or phage DNA template). Once isolated, the DNAcan be placed into expression vectors, which are then transfected intohost cells such as E. coli cells, simian COS cells, Chinese hamsterovary (CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of the desiredmonoclonal antibodies in the recombinant host cells.

DNA encoding the Fv clones of the invention can be combined with knownDNA sequences encoding heavy chain and/or light chain constant regions(e.g. the appropriate DNA sequences can be obtained from Kabat et al.,supra) to form clones encoding full or partial length heavy and/or lightchains. It will be appreciated that constant regions of any isotype canbe used for this purpose, including IgG, IgM, IgA, IgD, and IgE constantregions, and that such constant regions can be obtained from any humanor animal species. A Fv clone derived from the variable domain DNA ofone animal (such as human) species and then fused to constant region DNAof another animal species to form coding sequence(s) for “hybrid,” fulllength heavy chain and/or light chain is included in the definition of“chimeric” and “hybrid” antibody as used herein. In a preferredembodiment, a Fv clone derived from human variable DNA is fused to humanconstant region DNA to form coding sequence(s) for all human, full orpartial length heavy and/or light chains.

DNA encoding anti-CTLA4 or anti-PD-L1 antibody derived from a hybridomacan also be modified, for example, by substituting the coding sequencefor human heavy- and light-chain constant domains in place of homologousmurine sequences derived from the hybridoma clone. DNA encoding ahybridoma or Fv clone-derived antibody or fragment can be furthermodified by covalently joining to the immunoglobulin coding sequence allor part of the coding sequence for a non-immunoglobulin polypeptide. Inthis manner, “chimeric” or “hybrid” antibodies are prepared that havethe binding specificity of the Fv clone or hybridoma clone-derivedantibodies of the invention.

2. Antibody Fragments

In some embodiments, methods and compositions encompass antibodyfragments. In certain circumstances there are advantages of usingantibody fragments, rather than whole antibodies. The smaller size ofthe fragments may allow for rapid clearance, and may lead to improvedaccess to solid tumors.

Non-limiting examples of antibody fragments include Fab, Fab′, Fab′-SHand F(ab′)2 fragments of the anti-CTLA4 or anti-PD-L1 antibodiesprovided herein. These antibody fragments can be created by traditionalmeans, such as enzymatic digestion, or may be generated by recombinanttechniques. Some antibody fragments may be chimeric or humanized. Thesefragments are useful for the diagnostic and therapeutic purposes setforth below.

Various techniques may be used for the production of antibody fragments.Traditionally, these fragments were derived via proteolytic digestion ofintact antibodies, such as with pepsin or papain and/or by cleavage ofdisulfide bonds by chemical reduction. However, these fragments can nowbe produced directly by recombinant host cells. For example, Fab, Fv andScFv antibody fragments can all be expressed in and secreted from E.coli, thus allowing the facile production of large amounts of thesefragments. Alternatively, monoclonal antibody fragments can besynthesized using an automated peptide synthesizer.

Antibody fragments can be isolated from the antibody phage librariesdiscussed above. Alternatively, Fab′-SH fragments can be directlyrecovered from E. coli and chemically coupled to form F(ab′)2 fragments.According to another approach, F(ab′)2 fragments can be isolateddirectly from recombinant host cell culture. Other techniques for theproduction of antibody fragments will be apparent to the skilledpractitioner. In other embodiments, the antibody of choice is a singlechain Fv fragment (scFv). Fv and sFv are the only species with intactcombining sites that are devoid of constant regions; thus, they aresuitable for reduced nonspecific binding during in vivo use. sFv fusionproteins may be constructed to yield fusion of an effector protein ateither the amino or the carboxy terminus of an sFv. The antibodyfragment may also be a “linear antibody.” Such linear antibody fragmentsmay be monospecific or bispecific.

3. Humanized Antibodies

Some embodiments also encompass humanized antibodies. Various methodsfor humanizing non-human antibodies are known in the art. For example, ahumanized antibody can have one or more amino acid residues introducedinto it from a source which is non-human. These non-human amino acidresidues are often referred to as “import” residues, which are typicallytaken from an “import” variable domain. Humanization can be essentiallyperformed using any method known to those of ordinary skill in the art.Accordingly, such “humanized” antibodies are chimeric antibodies whereinsubstantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species. Inpractice, humanized antibodies are typically human antibodies in whichsome hypervariable region residues.

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to one method, humanized antibodies areprepared by a process of analysis of the parental sequences and variousconceptual humanized products using three-dimensional models of theparental and humanized sequences. Three-dimensional immunoglobulinmodels are commonly available and are familiar to those skilled in theart. Computer programs are available which illustrate and displayprobable three-dimensional conformational structures of selectedcandidate immunoglobulin sequences. Inspection of these displays permitsanalysis of the likely role of the residues in the functioning of thecandidate immunoglobulin sequence, i.e., the analysis of residues thatinfluence the ability of the candidate immunoglobulin to bind itsantigen. In this way, FR residues can be selected and combined from therecipient and import sequences so that the desired antibodycharacteristic, such as increased affinity for the target antigen(s), isachieved. In general, the hypervariable region residues are directly andmost substantially involved in influencing antigen binding.

4. Human Antibodies

Human anti-CTLA4, anti-PD-1, or anti-PD-L1 antibodies (or fragmentsthereof) can be constructed by combining Fv clone variable domainsequence(s) selected from human-derived phage display libraries withknown human constant domain sequences(s) as described above.Alternatively, human monoclonal anti-CTLA4, anti-PD-1, or anti-PD-L1antibodies can be made by the hybridoma method. Other methods known tothose of ordinary skill in the art can be utilized.

Transgenic animals (e.g. mice) can be produced that are capable, uponimmunization, of producing a full repertoire of human antibodies in theabsence of endogenous immunoglobulin production. For example, it hasbeen described that the homozygous deletion of the antibody heavy-chainjoining region (JH) gene in chimeric and germ-line mutant mice resultsin complete inhibition of endogenous antibody production. Transfer ofthe human germ-line immunoglobulin gene array in such germ-line mutantmice will result in the production of human antibodies upon antigenchallenge.

Gene shuffling can also be used to derive human antibodies fromnon-human, e.g. rodent, antibodies, where the human antibody has similaraffinities and specificities to the starting non-human antibody.According to one method, which is also called “epitope imprinting,”either the heavy or light chain variable region of a non-human antibodyfragment obtained by phage display techniques as described above isreplaced with a repertoire of human V domain genes, creating apopulation of non-human chain/human chain scFv or Fab chimeras.Selection with antigen results in isolation of a non-human chain/humanchain chimeric scFv or Fab wherein the human chain restores the antigenbinding site destroyed upon removal of the corresponding non-human chainin the primary phage display clone, i.e. the epitope governs (imprints)the choice of the human chain partner. When the process is repeated inorder to replace the remaining non-human chain, a human antibody isobtained. Unlike traditional humanization of non-human antibodies by CDRgrafting, this technique provides completely human antibodies, whichhave no FR or CDR residues of non-human origin.

5. Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is forCTLA4, PD-L1, or PD-1 and the other is for any other antigen. In someembodiments, one of the binding specificities is for CTLA4 and the otheris for PD-L1. Exemplary bispecific antibodies may bind to two differentepitopes of the CTLA4, PD-L1, or PD-1 protein. Bispecific antibodies mayalso be used to localize cytotoxic agents to cells which express CTLA4,PD-L1, or PD-1. These antibodies possess a CTLA4, PD-L1, or PD-1-bindingarm and an arm which binds the cytotoxic agent (e.g. saporin,anti-interferon-α, vinca alkaloid, ricin A chain, methotrexate orradioactive isotope hapten). Bispecific antibodies can be prepared asfull length antibodies or antibody fragments (e.g. F(ab′)2 bispecificantibodies).

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy chain-light chainpairs, where the two heavy chains have different specificities.

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion preferablyis with an immunoglobulin heavy chain constant domain, comprising atleast part of the hinge, CH2, and CH3 regions. It is preferred to havethe first heavy-chain constant region (CH1), containing the sitenecessary for light chain binding, present in at least one of thefusions. DNAs encoding the immunoglobulin heavy chain fusions and, ifdesired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable hostorganism. This provides for great flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yields. It is, however, possible to insert thecoding sequences for two or all three polypeptide chains in oneexpression vector when the expression of at least two polypeptide chainsin equal ratios results in high yields or when the ratios are of noparticular significance.

In some embodiments, the bispecific antibodies are composed of a hybridimmunoglobulin heavy chain with a first binding specificity in one arm,and a hybrid immunoglobulin heavy chain-light chain pair (providing asecond binding specificity) in the other arm. According to anotherapproach, the interface between a pair of antibody molecules can beengineered to maximize the percentage of heterodimers which arerecovered from recombinant cell culture.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. The “diabody” technology provides an alternativemechanism for making bispecific antibody fragments. The fragmentscomprise a heavy-chain variable domain (VH) connected to a light-chainvariable domain (VL) by a linker which is too short to allow pairingbetween the two domains on the same chain. Accordingly, the VH and VLdomains of one fragment are forced to pair with the complementary VL andVH domains of another fragment, thereby forming two antigen-bindingsites. Another strategy for making bispecific antibody fragmentsinvolves the use of single-chain Fv (sFv) dimmers.

6. Multivalent Antibodies

A multivalent antibody may be internalized (and/or catabolized) fasterthan a bivalent antibody by a cell expressing an antigen to which theantibodies bind. Antibodies can be multivalent antibodies (which areother than of the IgM class) with three or more antigen binding sites(e.g. tetravalent antibodies), which can be readily produced byrecombinant expression of nucleic acid encoding the polypeptide chainsof the antibody. The multivalent antibody may comprise a dimerizationdomain and three or more antigen binding sites. The preferreddimerization domain comprises (or consists of) an Fc region or a hingeregion. In this scenario, the antibody will comprise an Fc region andthree or more antigen binding sites amino-terminal to the Fc region. Insome embodiments, the multivalent antibody comprises (or consists of)three to about eight, but preferably four, antigen binding sites. Themultivalent antibody comprises at least one polypeptide chain (andpreferably two polypeptide chains), wherein the polypeptide chain(s)comprise two or more variable domains.

7. Antibody Variants

In some embodiments, amino acid sequence modification(s) of theantibodies described herein are contemplated. For example, it may bedesirable to improve the binding affinity and/or other biologicalproperties of the antibody. Amino acid sequence variants of the antibodyare prepared by introducing appropriate nucleotide changes into theantibody nucleic acid, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of, residues within the amino acid sequences of theantibody. Any combination of deletion, insertion, and substitution ismade to arrive at the final construct, provided that the final constructpossesses the desired characteristics. The amino acid alterations may beintroduced in the subject antibody amino acid sequence at the time thatsequence is made.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Non-limiting examples of terminalinsertions include an antibody with an N-terminal methionyl residue orthe antibody fused to a cytotoxic polypeptide. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody to an enzyme or a polypeptide which increasesthe serum half-life of the antibody or to a therapeutic amino acidsequence such as a thrombogenic polypeptide.

Another type of amino acid variant of the antibody alters the originalglycosylation pattern of the antibody. Such altering includes deletingone or more carbohydrate moieties found in the antibody, and/or addingone or more glycosylation sites that are not present in the antibody.Glycosylation of polypeptides is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used. Addition of glycosylation sites to theantibody is conveniently accomplished by altering the amino acidsequence such that it contains one or more of the above-describedtripeptide sequences (for N-linked glycosylation sites). The alterationmay also be made by the addition of, or substitution by, one or moreserine or threonine residues to the sequence of the original antibody(for O-linked glycosylation sites).

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the antibody moleculereplaced by a different residue. The sites of greatest interest forsubstitutional mutagenesis include the hypervariable regions, but FRalterations are also contemplated. Substantial modifications in thebiological properties of the antibody are accomplished by selectingsubstitutions that differ significantly in their effect on maintaining(a) the structure of the polypeptide backbone in the area of thesubstitution, for example, as a sheet or helical conformation, (b) thecharge or hydrophobicity of the molecule at the target site, or (c) thebulk of the side chain.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g. a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther development will have improved biological properties relative tothe parent antibody from which they are generated. A convenient way forgenerating such substitutional variants involves affinity maturationusing phage display. Briefly, several hypervariable region sites aremutated to generate all possible amino acid substitutions at each site.The antibodies thus generated are displayed from filamentous phageparticles as fusions to the gene III product of M13 packaged within eachparticle. The phage-displayed variants are then screened for theirbiological activity (e.g. binding affinity). In order to identifycandidate hypervariable region sites for modification, alanine scanningmutagenesis can be performed to identify hypervariable region residuescontributing significantly to antigen binding. Alternatively, oradditionally, it may be beneficial to analyze a crystal structure of theantigen-antibody complex to identify contact points between the antibodyand antigen. Such contact residues and neighboring residues arecandidates for substitution according to the techniques elaboratedherein. Once such variants are generated, the panel of variants issubjected to screening as described herein and antibodies with superiorproperties in one or more relevant assays may be selected for furtherdevelopment.

Nucleic acid molecules encoding amino acid sequence variants of theantibody are prepared by a variety of methods known in the art. Thesemethods include, but are not limited to, isolation from a natural source(in the case of naturally occurring amino acid sequence variants) orpreparation by oligonucleotide-mediated (or site-directed) mutagenesis,PCR mutagenesis, and cassette mutagenesis of an earlier prepared variantor a non-variant version of the antibody.

It may be desirable to introduce one or more amino acid modifications inan Fc region of the immunoglobulin polypeptides, thereby generating a Fcregion variant. In accordance with this description and the teachings ofthe art, it is contemplated that in some embodiments, an antibody usedin methods may comprise one or more alterations as compared to the wildtype counterpart antibody, e.g. in the Fc region. These antibodies wouldnonetheless retain substantially the same characteristics required fortherapeutic utility as compared to their wild type counterpart.

8. Antibody Derivatives

The antibodies described herein can be further modified to containadditional non-proteinaceous moieties that are known in the art andreadily available. For example, in some embodiments, the moietiessuitable for derivatization of the antibody are water soluble polymers.Non-limiting examples of water soluble polymers include, but are notlimited to, polyethylene glycol (PEG), copolymers of ethyleneglycol/propylene glycol, carboxymethylcellulose, dextran, polyvinylalcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane,ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymersor random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water. The polymer may be of any molecularweight, and may be branched or unbranched. The number of polymersattached to the antibody may vary, and if more than one polymers areattached, they can be the same or different molecules. In general, thenumber and/or type of polymers used for derivatization can be determinedbased on considerations including, but not limited to, the particularproperties or functions of the antibody to be improved, whether theantibody derivative will be used in a therapy under defined conditions,etc.

In these contexts, one may to link such antibodies to diagnostic ortherapeutic agents, or use them as capture agents or competitors incompetitive assays.

F. Pharmaceutical Compositions and Methods

In some embodiments, pharmaceutical compositions are administered to asubject. Different aspects involve administering an effective amount ofa composition to a subject. In some embodiments, a compositioncomprising an inhibitor may be administered to the subject or patient totreat cancer or reduce the size of a tumor. Additionally, such compoundscan be administered in combination with an additional cancer therapy.

Inhibitors can be formulated for parenteral administration, e.g.,formulated for injection via the intravenous, intramuscular,sub-cutaneous, or even intraperitoneal routes. Typically, suchcompositions can be prepared as injectables, either as liquid solutionsor suspensions; solid forms suitable for use to prepare solutions orsuspensions upon the addition of a liquid prior to injection can also beprepared; and, the preparations can also be emulsified. In addition tothe compounds formulated for parenteral administration, otherpharmaceutically acceptable forms include, e.g., aerosolizable,inhalable, or instillable formulations; tablets or other solids for oraladministration; time release capsules; creams; lotions; mouthwashes; andthe like. The preparation of such formulations will be known to those ofskill in the art in light of the present disclosure.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions; formulations including sesame oil,peanut oil, or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that it may be easily injected. It also should be stableunder the conditions of manufacture and storage and must be preservedagainst the contaminating action of microorganisms, such as bacteria andfungi.

The carrier also can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion, and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques, which yield a powder of the active ingredient, plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

As used herein, the term “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for contact withthe tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem complicationscommensurate with a reasonable benefit/risk ratio. The term“pharmaceutically acceptable carrier,” means a pharmaceuticallyacceptable material, composition or vehicle, such as a liquid or solidfiller, diluent, excipient, solvent or encapsulating material, involvedin carrying or transporting a chemical agent.

As used herein, “pharmaceutically acceptable salts” refers toderivatives of the disclosed compounds wherein the parent compound ismodified by converting an existing acid or base moiety to its salt form.Examples of pharmaceutically acceptable salts include, but are notlimited to, mineral or organic acid salts of basic residues such asamines; alkali or organic salts of acidic residues such as carboxylicacids; and the like. Pharmaceutically acceptable salts include theconventional non-toxic salts or the quaternary ammonium salts of theparent compound formed, for example, from non-toxic inorganic or organicacids. The pharmaceutically acceptable salts can be synthesized from theparent compound which contains a basic or acidic moiety by conventionalchemical methods.

Some variation in dosage will necessarily occur depending on thecondition of the subject. The person responsible for administrationwill, in any event, determine the appropriate dose for the individualsubject. An effective amount of therapeutic or prophylactic compositionis determined based on the intended goal. The term “unit dose” or“dosage” refers to physically discrete units suitable for use in asubject, each unit containing a predetermined quantity of thecomposition calculated to produce the desired responses discussed abovein association with its administration, i.e., the appropriate route andregimen. The quantity to be administered, both according to number oftreatments and unit dose, depends on the effects desired. Preciseamounts of the composition also depend on the judgment of thepractitioner and are peculiar to each individual. Factors affecting doseinclude physical and clinical state of the subject, route ofadministration, intended goal of treatment (alleviation of symptomsversus cure), and potency, stability, and toxicity of the particularcomposition.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeutically orprophylactically effective. The formulations are easily administered ina variety of dosage forms, such as the type of injectable solutionsdescribed above.

Typically, for a human adult (weighing approximately 70 kilograms), fromabout 0.1 mg to about 3000 mg (including all values and ranges therebetween), or from about 5 mg to about 1000 mg (including all values andranges there between), or from about 10 mg to about 100 mg (includingall values and ranges there between), of a compound are administered. Itis understood that these dosage ranges are by way of example only, andthat administration can be adjusted depending on the factors known tothe skilled artisan.

In certain embodiments, a subject is administered about, at least about,or at most about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09,0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4,1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8,2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7. 3.8, 3.9, 4.0, 4.1, 4.2,4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6,5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0,7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4,8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8,9.9, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0,15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0. 19.5, 20.0, 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150,155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220,225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290,295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360,365, 370, 375, 380, 385, 390, 395, 400, 410, 420, 425, 430, 440, 441,450, 460, 470, 475, 480, 490, 500, 510, 520, 525, 530, 540, 550, 560,570, 575, 580, 590, 600, 610, 620, 625, 630, 640, 650, 660, 670, 675,680, 690, 700, 710, 720, 725, 730, 740, 750, 760, 770, 775, 780, 790,800, 810, 820, 825, 830, 840, 850, 860, 870, 875, 880, 890, 900, 910,920, 925, 930, 940, 950, 960, 970, 975, 980, 990, 1000, 1100, 1200,1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400,2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600,3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800,4900, 5000, 6000, 7000, 8000, 9000, 10000 milligrams (mg) or micrograms(mcg) or μg/kg or micrograms/kg/minute or mg/kg/min ormicrograms/kg/hour or mg/kg/hour, or any range derivable therein. Inspecific embodiments, 50 mg/10 mL (5 mg/mL) of the inhibitor ipilimumabis administered. In specific embodiments, 200 mg/40 mL (5 mg/mL) of theinhibitor ipilimumab is administered.

A dose may be administered on an as needed basis or every 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 18, or 24 hours (or any range derivable therein)or 1, 2, 3, 4, 5, 6, 7, 8, 9, or times per day (or any range derivabletherein). A dose may be first administered before or after signs of aninfection are exhibited or felt by a patient or after a clinicianevaluates the patient for an infection. In some embodiments, the patientis administered a first dose of a regimen 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12 hours (or any range derivable therein) or 1, 2, 3, 4, or 5 daysafter the patient experiences or exhibits signs or symptoms of aninfection (or any range derivable therein). The patient may be treatedfor 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days (or any range derivabletherein) or until symptoms of an infection have disappeared or beenreduced or after 6, 12, 18, or 24 hours or 1, 2, 3, 4, or 5 days aftersymptoms of an infection have disappeared or been reduced. In specificembodiments, the inhibitor ipilimumab is administered every three weeks.

“Tumor,” as used herein, refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues. The terms “cancer,” “cancerous,” “cellproliferative disorder,” “proliferative disorder,” and “tumor” are notmutually exclusive as referred to herein.

The cancers amenable for treatment include, but are not limited to,melanoma, carcinoma, lymphoma, blastoma, sarcoma, and leukemia orlymphoid malignancies. More particular examples of such cancers includebreast cancer, colon cancer, rectal cancer, colorectal cancer, kidney orrenal cancer, clear cell cancer lung cancer including small-cell lungcancer, non-small cell lung cancer, adenocarcinoma of the lung andsquamous carcinoma of the lung, squamous cell cancer (e.g. epithelialsquamous cell cancer), cervical cancer, ovarian cancer, prostate cancer,prostatic neoplasms, liver cancer, bladder cancer, cancer of theperitoneum, hepatocellular cancer, gastric or stomach cancer includinggastrointestinal cancer, gastrointestinal stromal tumor, pancreaticcancer, head and neck cancer, glioblastoma, retinoblastoma, astrocytoma,thecomas, arrhenoblastomas, hepatoma, hematologic malignancies includingnon-Hodgkins lymphoma (NHL), multiple myeloma, myelodysplasticdisorders, myeloproliferative disorders, chronic myelogenous leukemia,and acute hematologic malignancies, endometrial or uterine carcinoma,endometriosis, endometrial stromal sarcoma, fibrosarcomas,choriocarcinoma, salivary gland carcinoma, vulval cancer, thyroidcancer, esophageal carcinomas, hepatic carcinoma, anal carcinoma, penilecarcinoma, nasopharyngeal carcinoma, laryngeal carcinomas, Kaposi'ssarcoma, mast cell sarcoma, ovarian sarcoma, uterine sarcoma, melanoma,malignant mesothelioma, skin carcinomas, Schwannoma, oligodendroglioma,neuroblastomas, neuroectodermal tumor, rhabdomyosarcoma, osteogenicsarcoma, leiomyosarcomas, Ewing Sarcoma, peripheral primitiveneuroectodermal tumor, urinary tract carcinomas, thyroid carcinomas,Wilm's tumor, as well as abnormal vascular proliferation associated withphakomatoses, edema (such as that associated with brain tumors), andMeigs' syndrome. In some cases, the cancer is melanoma. The cancerousconditions amenable for treatment include metastatic cancers.“Treatment” as used herein refers to clinical intervention in an attemptto alter the natural course of the individual or cell being treated, andcan be performed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include preventing occurrenceor recurrence of disease, alleviation of symptoms, reduction of anydirect or indirect pathological consequences of the disease, decreasingthe rate of disease progression, amelioration or palliation of thedisease state, and remission or improved prognosis. In some embodiments,antibodies are used to delay development of a disease or disorder. Innon-limiting examples, antibodies may be used to reduce the rate oftumor growth or reduce the risk of metastasis of a cancer.

“Treatment” as used herein refers to clinical intervention in an attemptto alter the natural course of the individual or cell being treated, andcan be performed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include preventing occurrenceor recurrence of disease, alleviation of symptoms, reduction of anydirect or indirect pathological consequences of the disease, decreasingthe rate of disease progression, amelioration or palliation of thedisease state, and remission or improved prognosis. In some embodiments,antibodies of the invention are used to delay development of a diseaseor disorder. In non-limiting examples, antibodies may be used to reducethe rate of tumor growth or reduce the risk of metastasis of a cancer.

Antibodies or antibody fragments can be used either alone or incombination with other compositions in a therapy. For instance, anantibody or antibody fragment may be co-administered withchemotherapeutic agent(s) (including cocktails of chemotherapeuticagents), other cytotoxic agent(s), anti-angiogenic agent(s), cytokines,thrombotic agents, and/or growth inhibitory agent(s). Such combinedtherapies noted above include combined administration (where the two ormore agents are included in the same or separate formulations), andseparate administration, in which case, administration of the antibodycan occur prior to, and/or following, administration of the adjuncttherapy or therapies.

Combination therapy may be achieved by use of a single pharmaceuticalcomposition that includes both agents, or by administering two distinctcompositions at the same time, wherein one composition includes theantibody and the other includes the second agent(s).

The two therapies may be given in either order and may precede or followthe other treatment by intervals ranging from minutes to weeks. Inembodiments where the other agents are applied separately, one wouldgenerally ensure that a significant period of time did not expirebetween the time of each delivery, such that the agents would still beable to exert an advantageously combined effect on the patient. In suchinstances, it is contemplated that one may administer both modalitieswithin about 12-24 h of each other and, more preferably, within about6-12 h of each other. In some situations, it may be desirable to extendthe time period for treatment significantly, however, where several d(2, 3, 4, 5, 6 or 7) to several wk (1, 2, 3, 4, 5, 6, 7 or 8) lapsebetween the respective administrations.

The antibodies and antibody fragments may also be administered incombination with radiotherapy, surgical therapy, immunotherapy(particularly radioimmunotherapy), gene therapy, or any other therapyknown to those of ordinary skill in the art for treatment of a diseaseor disorder associated with vascular proliferation, such as any of thediseases or disorders discussed elsewhere in this specification.

G. Examples

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

In Vivo Tumor Growth Assessment

6-8 week old C57BL/6 were purchased form Taconic and used 5-7 days afterarrival. On day 0 of the experimental protocol 2×10⁶ B16-SIY-dsRed cells(expressing the model peptide SIYRYYGL (SEQ ID NO.: 1)) were injectedsubcutaneously (s.c.) into the right flank. Treatment regimens wereinitiated on day 4, post tumor inoculation, and carried out through day16. Tumor growth was recorded every other day starting on day 7. Micewere sacrificed when either the tumor diameter reached 2 cm or theexperimental end-point of day 35 was reached. Immune cells from tumorand spleen of were analyzed for the immune status by flow cytometry andELISpot. See FIGS. 2-5.

TABLE 1 Drug Days of administration Concentration Route Clone CompanyAnti-CTLA4 Days 4, 7, 10 100 μg/dose i.p. UC10-4F10-11 BioXcellAnti-PD-L1 Every other day (4-16) 100 mg/dose i.p. 10F.9G2 BioXcellAnti-CD25 Day 4 100 mg/dose i.p. PC-61.5.3 BioXcell IL-7 Every day(4-15)  8 ng/dose s.c. (left flank) Peprotech IDOi Days 4-8 and 11-15300 mg/g QD Oral gavage IncyteFlow Cytometry

For flow cytometry analysis, spleens and tumors were isolated from mice,smashed, and single cell suspensions were prepared. Lymphocytes fromtumor samples were enriched via a ficoll (BD) centrifugation step (450g, 10 min., no break), and interphases were harvested and washed beforeused for staining procedure. Cells were stained with surface antibodiesand multimer for 45 min in FACS Buffer at 4° C. (PBS, 10% FCS, 0.5 mMEDTA). Following staining with multimer, cells were fixed in 4% PFA (BD)for 30 minutes and kept until acquisition in 1% PFA. Samples stained forT cell markers were fixed using a FoxP3 staining kit (eBioscience) andstained for FoxP3 according to manufacturer's instructions. Samples wereacquired on the LSRII blue machine (BD) and the data was analyzed usingFlowJo (TrekStar). The results are shown in FIG. 6.

TABLE 2 Clone Conjugate Company Staining panel T cell marker CD3 17A2AlexaF 700 eBioscience CD4 RM4-5 PerCP-Cy5.5 BioLegend CD8 53-6.7APC-Cy7 BioLegend CD44 IM7 FITC BD FoxP3 FJK-16a APC eBioscienceLive/death dye PacificBlue eBioscience Multimer panel CD3 17A2 AlexaF700 eBioscience CD4 RM4-5 PerCP-Cy5.5 BioLegend CD8 53-6.7 APC-Cy7BioLegend CD45/B220 RA3-6B2 FITC PharMingen SIY Pentamer PE ProimmuneLive/death dye PacificBlue eBioscienceELISpot.

For IFN-γ ELISpot (BD) analysis splenocytes were cryopreserved at a cellconcentration of 2×10⁷ cells/ml, collected until all mice from oneexperiments were available for analysis and thawed directly beforeinitializing the assay. In the assay 2×10⁶ cells were used per well andspecific stimulation was performed using the SIY peptide (160 nM) loadedonto splenocytes directly. Medium only served as negative control whilePMA/Ionomycin (2.5 μg/ml and 25 μg/ml final concentration, Sigma)stimulation was carried out as positive control. The assay itself wasperformed according to manufacturer's instructions and stimulation wasperformed for an 18 h period. The results are shown in FIG. 7.

Treatment Regimens.

Treatment was initiated on day 4 post tumor inoculation with thefollowing regimens for each drug. αCTLA-4 antibody (clone UC10-4F10-11,Bio-X-Cell) was given i.p. on day 4, 7 and 10 at a dose of 100 μg/mouse.αPD-L1 antibody (clone 10F.9G2, Bio-X-Cell) was given i.p. (100μg/mouse) every other day starting on day 4 ending on day 16 post tumorinoculation. IDOi (INCB23843, Incyte Corporation) was dissolved in 0.5%methylcellulose and administered at 300 mg/kg po QD on a 5 days on/2days off schedule starting on day 4. In the case of functionalexperiments with an earlier endpoint, treatment regimens were carriedout as described until the day of T cell analysis. A comparison betweenIDOi and IDOc (INCB24360, Incyte Corporation) was perfomed according tothe treatment regime described above. Tumor outgrowth was followedstarting day 7 onwards and is depicted in combination with anti-CTLA4(FIG. 18A) or anti-PD-L1 (FIG. 18B). N=5 mice per group, and a two-wayAnova test was used to determine significance between the groups.

Flow Cytometry and Antibodies.

For flow cytometric analysis, spleen, tumor-draining lymph node (TdLN),and tumor tissues were harvested at the indicated time point or whentumors reached a volume of 200 mm². Single cell suspensions wereprepared and a Ficoll-Hypaque purification step was performed for thetumor-derived cell suspension. Following a washing step, approximately2×10⁶ cells were used for antibody staining. Antibodies against thefollowing molecules were used if not otherwise indicated: CD3 (AF700,17A2, eBioscience), CD4 (PerCP-Cy5.5, RM4-5, Biolegend), CD8 (APCCy7,53-6.7, Biolegend), FoxP3 (APC, FJK-16a, eBioscience), IL-2 (PerCP,JES6-5H4, eBioscience), IFN-γ (APC, XMG1.2, eBioscience), and TNF-α(FITC, MP6-XT22, eBioscience). Fixable life/death discrimination wasperformed using PacBlue or AmCyan (eBioscience). Staining was carriedout at RT for 30 min if not indicated differently and intracellularstaining was performed using the FoxP3-staining kit according tomanufacturer's instructions (BD).

Staining of SIY-specific cells was performed using the SIYRYYGL-pentamer(Proimmune), conjugated with Phycoerythrin (PE), or as a non-specificcontrol with the SIINFEKL-pentamer. For staining, pentamers were diluted1:50 in PBS+10% FCS and incubated for 20 minutes at room temperature(RT). Following a washing step, cells were stained with specificantibodies for 30 minutes on ice prior to fixation in 4% PFA. All flowcytometric analyses were done using an LSR II blue instrument (BD) andanalyzed using FlowJo software (Tree Star).

Ex Vivo T Cell Functional Assays.

Single cell suspensions from tumor, spleen, and TdLN were prepared asdescribed above. Cell numbers were determined and cells were labeledwith Cell Trace (BD) according to manufacturer's instructions. A maximumof 1×10⁶ cells was plated per well on either non-treated or anti-CD3mAb-coated plates. Anti-CD3 mAb coating was performed with a solution of10 μg/ml αCD3 antibody (145-2C11, Biolegend) in PBS, incubated overnightat 4° C. Following 48 h of incubation, cells were harvested andtransferred onto newly anti-CD3-coated or non-treated plates, along withanti-CD28 mAb (2 μg/ml) (EL-4, Biolegend). Medium for all wells included5 μg/ml BrefeldinA (Sigma). Following a 6 h incubation at 37° C., cellswere harvested and stained for surface markers and intracellularcytokines using the technique described above.

Treatment with FTY720.

Prior to the initiation of the therapy regimens (2.5 h pre-treatment),fingolimod (FTY-720, Enzo Life Sciences) was given to mice to inhibitlymphocyte migration out of lymphoid organs. FTY-720 stock solution (10mg/ml in DMSO) was diluted to a 125 μg/ml concentration in PBS directlybefore administration. Mice received a dose of 25 μg FTY-720 or PBScontaining DMSO as control via oral gavage. Therapy was initiated thesame day (2.5 h delayed) and mice were analyzed on day 7 to perform theex vivo functional assay as described above.

In Vivo Proliferation Assay.

Assessment of in vivo proliferation was performed by Bromodeoxyuridine(BrdU) pulse in vivo, 24 h prior to flow cytometric analysis. Each mousereceived 0.8 mg BrdU in 100 μl injected i.p. either on day 6 or day 13of the treatment protocol. Mice were analyzed on day 7 or day 14,respectively, and cells were prepared for flow cytometry as describedabove. Following surface staining, cells were fixed using the FoxP3staining kit (BD). After the 30 minute fixation period, cells wereincubated in 100 μl of PBS/DNase solution (300 μg/ml) for 30 minutes at37° C. Cells were then washed and incubated for 30 minutes at RT withantibodies for FoxP3 and BrdU (FITC, Bu20a, eBioscience) followed byflow cytometric analysis.

Combinational Blockade of CTLA-4, PD-L1 or IDO Pathways Results inImproved Tumor Control In Vivo.

Single blockade of the immune-inhibitory pathways CTLA-4, PD-1/PD-L1, orIDO has been shown to have modest yet significant impact on tumor growthkinetics and to improve tumor-specific immune responses in various mousemodels in vivo. The effect of blocking multiple immune-inhibitorypathways on tumor control was investigated. On day 0, B16-SIY cells wereinoculated subcutaneously in the flank of C57BL/6 mice. After allowingthe tumor cells to engraft, therapy was initiated on day 4. Theanti-CTLA-4 mAb (clone: UC10-4F10-11) was given at three single timepoints. The anti-PD-L1 mAb (clone: 10F.9G2) was given every other daythroughout the treatment protocol, and the IDO inhibitor (IDOi,INCB23843) was given daily Monday-Friday via oral gavage. Treatment withsingle agents versus doublet combinations was compared, using tumorgrowth measured twice per week as the endpoint. For all three doubletreatments, improved tumor control was observed compared to thecorresponding single regimens. In particular, the combination of αCTLA-4and αPD-L1 resulted in 15 complete responders out of a total of 27treated mice (55.5%; FIG. 9B). Lower numbers of complete responders werefound for the combination of αCTLA-4 and IDOi (3/16; FIG. 9C) andimproved tumor control was also seen with αPD-L1 and IDOi. Together,these results suggest that combinatorial targeting ofCTLA-4+/−PD-L1+/−IDO could translate into a therapeutic advantage invivo.

Effective Combination Therapies do not Substantially Increase theFrequency of Anti-Tumor CD8⁺ T Cells in the Tumor-Draining Lymph Node atEarly Time Points.

The mechanism by which improved immune-mediated tumor control wasexamined. To test whether successful doublet therapies were firstimproving the de novo priming of anti-tumor T cells in thetumor-draining lymph node which then subsequently home to tumor sitesand improve tumor control, the frequency of SIY-specific CD8⁺ T cells inthe tumor-draining lymph node (TdLN) and in the spleen on day 7, usingSIY-K^(b) pentamer staining was assessed (FIGS. 10A-B). However, onlyminimal increases in frequencies of tumor-specific CD8⁺ T cells wereobserved. The functional capacity of these cells was further assessed byIFN-γ ELISpot (FIG. 10C). No major differences were detected betweentreatment groups. Only αCTLA-4+αPD-L1 and αCTLA-4+IDOi showed astatistically significant difference compared to no treatment (p=0.0263and p=0.0101) as well as to their respective single treatments(αCTLA-4+αPD-L to αPD-L1 p=0.0172; αCTLA-4+IDOi to αCTLA-4 p=0.0185).However, this difference did not seem sufficient to account for themajor improvement in tumor control observed.

Effective Doublets Result in Increased Frequency of IL-2-Producing andProliferating Polyfunctional T Cells within the Tumor.

In order to assess whether the therapeutic effect was a result ofreactivation of tumor-infiltrating CD8⁺ T cells, an ex vivo stimulationprotocol which is designed to favor analysis of pre-activated T cellswas utilized. Responsiveness was assessed by measuring proliferation(cell trace dilution) as well as production of IL-2, IFN-γ and TNF-α byintracellular cytokine staining. As depicted in a representative flowcytometric plot in FIG. 11A, only the therapeutic effective doublettreatments showed a detectable proliferation rate of CD8⁺ T cells incombination with IL-2 production, and the magnitude of this effect wasstriking (non-stimulated control shown in FIG. 12A). A statisticalanalysis of data spanning two independent experiments confirmed thatsignificant ex vivo proliferation was only observed in stimulated Tcells from mice that received the effective doublets (FIG. 11B). Modestincreases in both IFN-γ and TNF-α production were also seen; however,high production of these cytokines by tumor-infiltrating CD8⁺ T cellswas already observed without treatment. Looking further atIFN-γ-producing cells in the doublet treatment groups, mostIFN-γ-producing T cells were positive for IL-2 production and showedsignificantly increased proliferation (FIG. 11C), while single treatmentgroups showed mainly IFN-γ-single producing cells. Comparing thestimulated cells to non-stimulated controls confirmed that only thedouble treatments were able to show increased IL-2 production andproliferation above background levels (FIG. 12B). These T cells alsoproduced TNF-α, indicating a polyfunctional T cell phenotype (FIG. 11D).Detection of cytokine production was dependent on re-stimulation invitro (FIG. 12B). Thus, all three effective immunotherapy doubletsresulted in the same improved functional effect: restoration of IL-2production and proliferation by CD8⁺ intratumoral T cells, along withaugmented polyfunctionality.

Increased Frequency of Polyfunctional T Cells within the Tumor does notRequire New T Cell Migration.

To determine whether the presence of CD8⁺ T cells showing high levels ofIL-2 production and proliferation within the tumor microenvironmentfollowing effective immunotherapy doublets was a result of new T cellmigration into the tumor site or re-activation of T cells alreadypresent, FTY720 treatment was utilized to block the sphingosine1-phosphate receptor-1 and thereby prohibit T cells from exitinglymphoid organs. Previous studies have shown that the effect can bedetected as soon as 2 h after initial administration and is stable forup to 4 days. FTY720 was administered or control vehicle totumor-bearing mice on day 4, 2.5 h prior to the initiation ofimmunotherapies. To control for effective depletion of circulating Tcells the number of CD3⁺ cells in the peripheral blood was assesed.Overall, a 90% reduction in circulating CD3⁺ T cells in FTY720-treatedmice was detected (FIG. 13A).

The effect of intratumoral CD8⁺ T cells on IL-2 production andproliferation restoration was examined. Comparing vehicle-treated groups(open bars) to the FTY720-treated groups (filled bars) a similarincrease in proliferation and IL-2 production was observed (FIGS.13B-C), although there were some subtle differences. A modest reductionof proliferated/IL-2⁺-cells could be detected after αCTLA-4+IDOitreatment and a modest increase could be observed when mice were treatedwith αPD-L1+IDOi (p=0.0286) (FIG. 13C). Nevertheless, these datastrongly suggest that the major mechanism for improved T cell functionwithin the tumor microenvironment following these effectiveimmunotherapies is through a direct effect on CD8⁺ T cells alreadypresent in the tumor site.

Immunotherapy doublet therapy was employed to examine the effect onproliferation of tumor-infiltrating T cells. A short pulse of BrdUadministration in vivo was used. To this end, a single dose of BrdU wasadministered i.p. on day 6 and proliferation of T cells in the tumor(FIG. 14), spleen and TdLN (FIG. 15) was assessed on day 7. Consistentwith the ex vivo results, the doublet-treated mice harbored moreproliferating CD8⁺ T cells in the tumor site than did the single-treatedgroups or the mice that received no treatment (FIG. 14A). A similareffect was also seen for CD4⁺ T cells, with the exception that αCTLA4mAbsingle treatment also resulted in an increased proliferation rate inthis cell compartment (FIG. 14B). Taken together, these data indicatethat the therapeutically successful combination therapies ofanti-CTLA-4+/−anti-PD-L1+/−an IDOi resulted in increased proliferationand reactivation of CD8⁺ T cells directly within the tumor site.

Combinatorial Treatments Lead to Prolonged Persistence and HigherFrequency of Tumor-Reactive Lymphocytes in the Periphery at Later TimePoints.

To test if combinatorial treatment restored T cell function within thetumor site, and led to anti-tumor T cell recirculation in the periphery,functional SIY-specific T cells were assayed for in the spleen on day 14(FIG. 16). Indeed, a significant increase in the frequency ofIFN-γ-producing T cells upon SIY stimulation was observed when comparingthe double treatments to the single treatment or no treatment groups.All three of the double treatment groups show an increase inSIY-reactive cells by 2- to even 3.5-fold higher levels compared to day7. This effect was accompanied by increased frequency of SIY-pentamerpositive CD8⁺ T cells in the spleen, and also within the tumor (FIG.17). Consistent with these results, re-challenge of mice 6 weeks laterthat rejected the first tumor were protected against B16.SIY (Table 3).Thus, the successful doublet treatments eventually led to a highercirculating fraction of tumor antigen-specific T cells that likelyrepresents a memory response.

TABLE 3 # # complete # # protective treatment mice rejectionrechallenged response αCTLA-4 37 0 0 0 αPD-L1 31 0 0 0 IDOi 26 0 0 0αCTLA-4 + αPD-L1 27 15 5 4 αCTLA-4 + IDOi 16 3 3 2 αPD-L1 + IDOi 10 1 10 Mice with a complete rejection of the tumor after therapy wererechallenged with 2 × 10⁶ B16-SIY cells 4 weeks after therapy was ended.Tumor growth was followed up to 8 weeks after rechallengePairwise Combinations of αCTLA-4, αPD-L1 or IDOi Blockade Results inRetarded Tumor Outgrowth.

αCTLA-4 was administered on day 4,7,10 i.p., αPD-L1 on day4,6,8,10,12,14,16 i.p. and IDOi was given every day via oral gavageMonday-Friday. Tumor outgrowth measured in mm² comparing the singletreatment to the respective combined double treatment of αCTLA-4 andαPD-L1 C (FIG. 9A), αCTLA-4 and IDOi (FIG. 9B), and αPD-L1 and IDOi(FIG. 9C). Depicted are means+/−SEM of 6 mice from one representativeexperiment. All experiments were at least done twice with the sameoverall result. Significance to the single treatments was tested using atwo-way-Anova with Bonferroni post-test and is shown in the figure whileall treatments regimes were significantly different to the no treatmentcontrol (****<0.0001, **<0.01).

Pairwise Combinations of αCTLA-4 or αPD-L1 with Either IDOi (INCB23843)or IDOc (INCB24360) Blockade Results in Similar Tumor OutgrowthRetardation.

Treatment was administered in the same fashion as described for FIG. 9,with the exception that IDOi (INCB23843) was replaced by IDOc(INCB24360) in the indicated groups. FIG. 18A shows combination of IDOiand IDOc with αCTLA-4 and FIG. 18B depicts combinations of IDOi and IDOcwith αPD-L1. Shown are means+/−SEM of 5 mice per group from arepresentative experiment and significance was determined using atwo-way-Anova with Bonferroni post-test (****<0.0001, ***<0.001,**<0.01, n.s.=not significantly different).

Unaffected Fraction Analysis of the Combination Therapies DemonstratesSynergy.

Synergy analysis was performed with day 15 data and with day 23 data.Actual tumor growth inhibition (TGI) is the experimental TGI seen inthis experiment for the combinations, and predicted TGI is the TGI thatwould have been expected for a purely additive combination effect fromthe performance of the individual agents in this experiment. Actual TGIshigher than predicted TGIs reflect better than additive (synergistic)data. Table 4. demonstrates that IDOi+αCTLA4, IDOi+αPDL1, IDOc+αCTLA4and IDOc+αPDL-1 are synergistic treatments.

TABLE 4 Day 23 (last day of Day 15 (end of dosing) vehicles) anti antiCTLA4 antiPDL1 CTLA4 antiPDL1 IDOc (INCB24360) Actual TGI 74% 81% 93%82% Predicted TGI 66% 24% 65% 35% IDOi (INCB23843) Actual TGI 75% 88%87% 78% Predicted TGI 69% 31% 67% 39%Double Regimen Therapy does not Result in Substantially IncreasedFrequency of Tumor-Reactive T Cells at Early Time Points in thePeriphery.

Peptide/K^(b) pentamer staining was performed on gated CD3+CD8+ T cells,isolated from TdLN (FIG. 10A) or spleen (FIG. 10B) on day 7. Shown aremeans+/−SEM of a total of 10 mice collected from two experiments. Forstatistical analysis, Mann-Whitney-U test was performed comparing singletreatments to double treatments (*=0.0317 αCTLA-4 to αCTLA-4+αPD-L1 inspleen). None of the other treatments was significantly different to theno treatment control. IFN-γ ELISpot was performed on splenocytescollected on day 7 (FIG. 10C). Data are shown as mean+/−SEM from 10 miceout of two experiments with no stimulation as open bar and SIY-specificstimulation as filled bar. The Mann-Whitney-U test was used to assesssignificant differences between no treatment group and treatmentregimens with * in the figure indicating significant difference comparedto no treatment. Results with αCTLA-4 were significantly different toαCTLA-4+IDOi (p=0.0185) and αPD-L1 was significantly different toαCTLA-4+αPD-L1 (p=0.0172).

Double Treatments Restore Capacity of Lymphocytes within the Tumor toProduce IL-2 and Proliferate.

Tumors were harvested on day 7 and single cell suspensions wereprepared. Pools of cells from 3-5 mice were combined and subsequentlystained with cell trace. Cells were cultured with or without plate-boundanti-CD3 for 48 h then treated with anti-CD3/anti-CD28-stimulation inthe presence of Brefeldin A for 6h. Cells were then stained forproduction of IL-2, IFN-γ and TNF-α by intracellular flow cytometry. Arepresentative FACS plot showing proliferation via cell trace dilutionon the x-axis and intracellular IL-2 staining on the y-axis (FIG. 11A).A pool of five mice was analyzed (FIG. 11B). Statistical analysis of theamount of proliferating CD3⁺CD8⁺ cells in the non-stimulated (left) andstimulated (right) group. Only double treatments show a significantincrease in proliferation compared to non-stimulated and singletreatments when tested with a one-way Anova. Bars represent mean+/−SEMof a total of 4 pools collected out of 2 experiments. The percentages ofIFN-γ⁺ (open bar), IFN-γ⁺ and IL-2⁺ (gray bar), and proliferatingIFN-γ⁺IL-2⁺ cells (filled bar) were calculated within the CD3⁺CD8⁺ cellpopulation (FIG. 11C).

Restoration of IL-2 Production and Proliferation of Tumor-InfiltratingLymphocytes in the Absence of New T Cell Migration.

B16-SIY bearing mice were either treated with FTY720 or control vehicleprior to initiation of therapy, to prevent migration of new lymphocytesinto the tumor. Peripheral blood T cell numbers following FTY720treatment on the day of tumor harvest for analysis. Open bars depict thenumber of CD45⁺CD3⁺ T cells detected in 200 ul peripheral blood ofvehicle treated mice set to 100%. Filled bars represent the number foundin FTY720-treated mice, relative to the vehicle-treated group (FIG.13A). Single cell suspensions from tumor were labeled with cell traceand stimulated with plate-bound anti-CD3 antibody for 48 h prior thenwith anti-CD3 and anti-CD28 in the presence of Brefeldin A. Cells werethen analyzed for proliferation by cell trace dilution and production ofIL-2 via intracellular staining. Depicted are the percentages ofproliferating cells (FIG. 13B) or proliferating and IL-2 producing cells(FIG. 13C) comparing vehicle-treated groups (open bar) to FTY720-treatedgroups (filled bar). Results are shown as the mean+/−SEM combining twoexperiments with each having 2 pools of 3 mice. Significance was testedusing Mann-Whitney-U test but no significant change between FTY720 andvehicle control could be detected except for increased IL-2 productionin the αPD-L1+IDOi treatment group (p=0.02).

Immunotherapy Doublets Result in Increased BrdU Incorporation by CD8+and CD4+ Tumor-Infiltrating T Cells In Vivo.

Tumor-infiltrating lymphocytes were harvested on day 7, 24 h after asingle BrdU pulse in vivo, and cells were stained for BrdU along withanti-CD3, anti-CD4, and anti-CD8. Depicted are percentages of BrdU⁺cells that were CD3⁺ CD8⁺ (FIG. 14A) and CD3⁺CD4⁺ (FIG. 14B). Data shownpresent the mean of a total of 5 mice from one experiment and arerepresentative of two independent experiments. Differences were assessedusing a two-way Anova test and taking proliferation values from spleenand TdLN into account. * indicates significantly different to notreatment and all double treatments were significantly different totheir corresponding single treatments with the exception of αCTLA-4 toboth double treatments for CD4 T cell proliferation.

In Vivo Proliferation of CD8+ and CD4+ Positive T Cells in Spleen andTdLN.

To contrast to the increased BrDU incorporation observed among T cellsin the tumor, BrdU staining was performed on cells from spleen and TdLNand analyzed side by side to the staining shown in FIGS. 14A-B. Barsrepresent the mean+/−SEM out of a total of 10 mice. No differences weresignificant. The results are shown in FIG. 15.

Immunotherapy Doublets Result in Increased Frequencies of TumorAntigen-Specific T Cells at Later Time Points in the Periphery.

Depicted is an IFN-γ ELISpot of splenocytes harvested on day 14 withopen bars being the un-stimulated control and filled bars representingSIY-stimulation. Data are shown as the mean+/−SEM from 10 mice pooledfrom two experiments. Statistical analysis was done using Mann-Whitney-Utest comparing all groups to no treatment. All double treatment groupswere significantly different to their respective single treatment andwhen comparing double treatments within each other a significantdifference between αCTLA4+αPD-L1 and αPD-L1+IDOi was observed. Theresults are shown in FIG. 16.

Immunotherapy Doublets Result in Increased Frequency and LongerPersistence of SIY/K^(b) Pentamer-Specific T Cells in the Periphery andin the Tumor.

Pentamer staining was performed on gated CD3⁺CD8⁺ T cells, isolated fromspleen, TdLN or tumor on day 14. Shown are means+/−SEM of a total of 10mice collected from two experiments. For statistical analysis,Mann-Whitney-U test was performed comparing all treatments to the notreatment control (indicated by *). The following combinations weresignificantly different in the spleen: αCTLA4 to αCTLA4+αPD-L1 andαCTLA4+IDOi; αPD-L1 to αCTLA4+αPD-L1 and αPD-L1+IDOi; IDOi toαCTLA4+IDOi; and in the tumor: αCTLA4 to αCTLA4+αPD-L1 and αCTLA4+IDOi;αPD-L1 to αCTLA4+αPD-L1; IDOi to αCTLA4+IDOi. The results are shown inFIG. 17.

Doublet therapies using either anti-CTLA-4, anti-PD-L1 and/or IDOi showa synergistic retardation of tumor outgrowth in vivo. The major biologiccorrelate to this improved efficacy was restored IL-2 production andproliferation of tumor-infiltrating CD8+ T cells. In addition, thisfunctional restoration does not require new T cell migration as assessedusing FTY720 administration. Successful combination immunotherapiesfunction, at least in part, by correcting functional defects of T cellsdirectly within the tumor microenvironment.

CD8+ TILs without any therapy showed significant production of IFN-γproduction when analyzed ex vivo. Consistent with this observation,human melanoma metastases showing a T cell-infiltrated phenotype usuallyshow expression of IFN-γ-induced target genes and in many cases IFN-γitself. IFN-γ produced by CD8+ T cells is necessary for the induction ofthe negative regulatory factors PD-L1 and IDO within the tumormicroenvironment. Thus, the retained ability of TIL to produce at leastsome IFN-γ may in fact contribute to the negative regulatory networkwithin the tumor site that enable tumor immune evasion.

Example 14-({2-[(Aminosulfonyl)amino]ethyl}amino)-N-(3-bromo-4-fluorophenyl)-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide

Step 1: 4-Amino-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide

Malononitrile (320.5 g, 5 mol) was added to water (7 L) preheated to 45°C. and stirred for 5 min. The resulting solution was cooled in an icebath and sodium nitrite (380 g, 5.5 mol) was added. When the temperaturereached 10° C., 6 N hydrochloric acid (55 mL) was added. A mildexothermic reaction ensued with the temperature reaching 16° C. After 15min the cold bath was removed and the reaction mixture was stirred for1.5 hrs at 16-18° C. The reaction mixture was cooled to 13° C. and 50%aqueous hydroxylamine (990 g, 15 mol) was added all at once. Thetemperature rose to 26° C. When the exothermic reaction subsided thecold bath was removed and stirring was continued for 1 hr at 26-27° C.,then it was slowly brought to reflux. Reflux was maintained for 2 hrsand then the reaction mixture was allowed to cool overnight. Thereaction mixture was stirred in an ice bath and 6 N hydrochloric acid(800 mL) was added in portions over 40 min to pH 7.0. Stirring wascontinued in the ice bath at 5° C. The precipitate was collected byfiltration, washed well with water and dried in a vacuum oven (50° C.)to give the desired product (644 g, 90%). LCMS for C₃H₆N₅O₂ (M+H)⁺:m/z=144.0. ¹³C NMR (75 MHz, CD₃OD): δ 156.0, 145.9, 141.3.

Step 2: 4-Amino-N-hydroxy-1,2,5-oxadiazole-3-carboximidoyl chloride

4-Amino-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide (422 g, 2.95 mol)was added to a mixture of water (5.9 L), acetic acid (3 L) and 6 Nhydrochloric acid (1.475 L, 3 eq.) and this suspension was stirred at42-45° C. until complete solution was achieved. Sodium chloride (518 g,3 eq.) was added and this solution was stirred in an ice/water/methanolbath. A solution of sodium nitrite (199.5 g, 0.98 eq.) in water (700 mL)was added over 3.5 hrs while maintaining the temperature below 0° C.After complete addition stirring was continued in the ice bath for 1.5hrs and then the reaction mixture was allowed to warm to 15° C. Theprecipitate was collected by filtration, washed well with water, takenin ethyl acetate (3.4 L), treated with anhydrous sodium sulfate (500 g)and stirred for 1 hr. This suspension was filtered through sodiumsulfate (200 g) and the filtrate was concentrated on a rotaryevaporator. The residue was dissolved in methyl t-butyl ether (5.5 L),treated with charcoal (40 g), stirred for 40 min and filtered throughCelite. The solvent was removed in a rotary evaporator and the resultingproduct was dried in a vacuum oven (45° C.) to give the desired product(256 g, 53.4%). LCMS for C₃H₄ClN₄O₂ (M+H)⁺: m/z=162.9. ¹³C NMR (100 MHz,CD₃OD): δ 155.8, 143.4, 129.7.

Step 3:4-Amino-N′-hydroxy-N-(2-methoxyethyl)-1,2,5-oxadiazole-3-carboximidamide

4-Amino-N-hydroxy-1,2,5-oxadiazole-3-carboximidoyl chloride (200.0 g,1.23 mol) was mixed with ethyl acetate (1.2 L). At 0-5° C.2-methoxyethylamine [Aldrich, product #143693] (119.0 mL, 1.35 mol) wasadded in one portion while stirring. The reaction temperature rose to41° C. The reaction was cooled to 0-5° C. Triethylamine (258 mL, 1.84mol) was added. After stirring 5 min, LCMS indicated reactioncompletion. The reaction solution was washed with water (500 mL) andbrine (500 mL), dried over sodium sulfate, and concentrated to give thedesired product (294 g, 119%) as a crude dark oil. LCMS for C₆H₁₂N₅O₃(M+H)⁺: m/z=202.3. ¹H NMR (400 MHz, DMSO-d₆): δ 10.65 (s, 1H), 6.27 (s,2H), 6.10 (t, J=6.5 Hz, 1H), 3.50 (m, 2H), 3.35 (d, J=5.8 Hz, 2H), 3.08(s, 3H).

Step 4:1V-Hydroxy-4-[(2-methoxyethyl)amino]-1,2,5-oxadiazole-3-carboximidamide

4-Amino-N′-hydroxy-N-(2-methoxyethyl)-1,2,5-oxadiazole-3-carboximidamide (248.0 g, 1.23 mol) was mixedwith water (1 L). Potassium hydroxide (210 g, 3.7 mol) was added. Thereaction was refluxed at 100° C. overnight (15 hours). TLC with 50%ethyl acetate (containing 1% ammonium hydroxide) in hexane indicatedreaction completed (product Rf=0.6, starting material Rf=0.5). LCMS alsoindicated reaction completion. The reaction was cooled to roomtemperature and extracted with ethyl acetate (3×1 L). The combined ethylacetate solution was dried over sodium sulfate and concentrated to givethe desired product (201 g, 81%) as a crude off-white solid. LCMS forC₆H₁₂N₅O₃ (M+H)⁺: m/z=202.3 ¹H NMR (400 MHz, DMSO-d₆): δ 10.54 (s, 1H),6.22 (s, 2H), 6.15 (t, J=5.8 Hz, 1H), 3.45 (t, J=5.3 Hz, 2H), 3.35 (m,2H), 3.22 (s, 3H).

Step 5:N-Hydroxy-4-[(2-methoxyethyl)amino]-1,2,5-oxadiazole-3-carboximidoylchloride

At room temperatureN′-hydroxy-4-[(2-methoxyethyl)amino]-1,2,5-oxadiazole-3-carboximidamide(50.0 g, 0.226 mol) was dissolved in 6.0 M hydrochloric acid aqueoussolution (250 mL, 1.5 mol). Sodium chloride (39.5 g, 0.676 mol) wasadded followed by water (250 mL) and ethyl acetate (250 mL). At 3-5° C.a previously prepared aqueous solution (100 mL) of sodium nitrite (15.0g, 0.217 mol) was added slowly over 1 hr. The reaction was stirred at3-8° C. for 2 hours and then room temperature over the weekend. LCMSindicated reaction completed. The reaction solution was extracted withethyl acetate (2×200 mL). The combined ethyl acetate solution was driedover sodium sulfate and concentrated to give the desired product (49.9g, 126%) as a crude white solid. LCMS for C₆H₁₀C₁N₄O₃ (M+H)⁺: m/z=221.0.¹H NMR (400 MHz, DMSO-d₆): δ 13.43 (s, 1H), 5.85 (t, J=5.6 Hz, 1H), 3.50(t, J=5.6 Hz, 2H), 3.37 (dd, J=10.8, 5.6 Hz, 2H), 3.25 (s, 3H).

Step 6:N-(3-Bromo-4-fluorophenyl)-N′-hydroxy-4-[(2-methoxyethyl)amino]-1,2,5-oxadiazole-3-carboximidamide

N-Hydroxy-4-[(2-methoxyethyl)amino]-1,2,5-oxadiazole-3-carboximidoylchloride (46.0 g, 0.208 mol) was mixed with water (300 mL). The mixturewas heated to 60° C. 3-Bromo-4-fluoroaniline [Oakwood products, product#013091] (43.6 g, 0.229 mol) was added and stirred for 10 min. A warmsodium bicarbonate (26.3 g, 0.313 mol) solution (300 mL water) was addedover 15 min. The reaction was stirred at 60° C. for 20 min. LCMSindicated reaction completion. The reaction solution was cooled to roomtemperature and extracted with ethyl acetate (2×300 mL). The combinedethyl acetate solution was dried over sodium sulfate and concentrated togive the desired product (76.7 g, 98%) as a crude brown solid. LCMS forC₁₂H₁₄BrFN₅O₃ (M+H)⁺: m/z=374.0, 376.0. ¹H NMR (400 MHz, DMSO-d₆): δ11.55 (s, 1H), 8.85 (s, 1H), 7.16 (t, J=8.8 Hz, 1H), 7.08 (dd, J=6.1,2.7 Hz, 1H), 6.75 (m, 1H), 6.14 (t, J=5.8 Hz, 1H), 3.48 (t, J=5.2 Hz,2H), 3.35 (dd, J=10.8, 5.6 Hz, 2H), 3.22 (s, 3H).

Step 7:4-(3-Bromo-4-fluorophenyl)-3-{4-[(2-methoxyethyl)amino]-1,2,5-oxadiazol-3-yl}-1,2,4-oxadiazol-5(4H)-one

A mixture ofN-(3-bromo-4-fluorophenyl)-N′-hydroxy-4-[(2-methoxyethyl)amino]-1,2,5-oxadiazole-3-carboximidamide(76.5 g, 0.204 mol), 1,1′-carbonyldiimidazole (49.7 g, 0.307 mol), andethyl acetate (720 mL) was heated to 60° C. and stirred for 20 min. LCMSindicated reaction completed. The reaction was cooled to roomtemperature, washed with 1 N HCl (2×750 mL), dried over sodium sulfate,and concentrated to give the desired product (80.4 g, 98%) as a crudebrown solid. LCMS for C₁₃H₁₂BrFN₅O₄ (M+H)⁺: m/z=400.0, 402.0. ¹H NMR(400 MHz, DMSO-d₆): δ 7.94 (t, J=8.2 Hz, 1H), 7.72 (dd, J=9.1, 2.3 Hz,1H), 7.42 (m, 1H), 6.42 (t, J=5.7 Hz, 1H), 3.46 (t, J=5.4 Hz, 2H), 3.36(t, J=5.8 Hz, 2H), 3.26 (s, 3H).

Step 8:4-(3-Bromo-4-fluorophenyl)-3-{4-[(2-hydroxyethyl)amino]-1,2,5-oxadiazol-3-yl}-1,2,4-oxadiazol-5(4H)-one

4-(3-Bromo-4-fluorophenyl)-3-{4-[(2-methoxyethyl)amino]-1,2,5-oxadiazol-3-yl}-1,2,4-oxadiazol-5(4H)-one(78.4 g, 0.196 mol) was dissolved in dichloromethane (600 mL). At −67°C. boron tribromide (37 mL, 0.392 mol) was added over 15 min. Thereaction was warmed up to −10° C. in 30 min. LCMS indicated reactioncompleted. The reaction was stirred at room temperature for 1 hour. At0-5° C. the reaction was slowly quenched with saturated sodiumbicarbonate solution (1.5 L) over 30 min. The reaction temperature roseto 25° C. The reaction was extracted with ethyl acetate (2×500 mL, firstextraction organic layer is on the bottom and second extraction organiclager is on the top). The combined organic layers were dried over sodiumsulfate and concentrated to give the desired product (75 g, 99%) as acrude brown solid. LCMS for C₁₂H₁₀BrFN₅O₄ (M+H)⁺: m/z=386.0, 388.0. ¹HNMR (400 MHz, DMSO-d₆): δ 8.08 (dd, J=6.2, 2.5 Hz, 1H), 7.70 (m, 1H),7.68 (t, J=8.7 Hz, 1H), 6.33 (t, J=5.6 Hz, 1H), 4.85 (t, J=5.0 Hz, 1H),3.56 (dd, J=10.6, 5.6 Hz, 2H), 3.29 (dd, J=11.5, 5.9 Hz, 2H).

Step 9:2-({4-[4-(3-Bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}amino)ethylmethanesulfonate

To a solution of4-(3-bromo-4-fluorophenyl)-3-{4-[(2-hydroxyethyl)amino]-1,2,5-oxadiazol-3-yl}-1,2,4-oxadiazol-5(4H)-one(1.5 kg, 3.9 mol, containing also some of the correspondingbromo-compound) in ethyl acetate (12 L) was added methanesulfonylchloride (185 mL, 2.4 mol) dropwise over 1 h at room temperature.Triethylamine (325 mL, 2.3 mol) was added dropwise over 45 min, duringwhich time the reaction temperature increased to 35° C. After 2 h, thereaction mixture was washed with water (5 L), brine (1 L), dried oversodium sulfate, combined with 3 more reactions of the same size, and thesolvents removed in vacuo to afford the desired product (7600 g,quantitative yield) as a tan solid. LCMS for C₁₃H₁₁BrFN₅O₆SNa (M+Na)⁺:m/z=485.9, 487.9. ¹H NMR (400 MHz, DMSO-d₆): δ 8.08 (dd, J=6.2, 2.5 Hz,1H), 7.72 (m, 1H), 7.58 (t, J=8.7 Hz, 1H), 6.75 (t, J=5.9 Hz, 1H), 4.36(t, J=5.3 Hz, 2H), 3.58 (dd, J=11.2, 5.6 Hz, 2H), 3.18 (s, 3H).

Step 10:3-{4-[(2-Azidoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-(3-bromo-4-fluorophenyl)-1,2,4-oxadiazol-5(4H)-one

To a solution of2-({4-[4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}amino)ethylmethanesulfonate (2.13 kg, 4.6 mol, containing also some of thecorresponding bromo-compound) in dimethylformamide (4 L) stirring in a22 L flask was added sodium azide (380 g, 5.84 mol). The reaction washeated at 50° C. for 6 h, poured into ice/water (8 L), and extractedwith 1:1 ethyl acetate:heptane (20 L). The organic layer was washed withwater (5 L) and brine (5 L), and the solvents removed in vacuo to affordthe desired product (1464 g, 77%) as a tan solid. LCMS forC₁₂H₈BrFN₈O₃Na (M+Na)⁺: m/z=433.0, 435.0. ¹H NMR (400 MHz, DMSO-d₆): δ8.08 (dd, J=6.2, 2.5 Hz, 1H), 7.72 (m, 1H), 7.58 (t, J=8.7 Hz, 1H), 6.75(t, J=5.7 Hz, 1H), 3.54 (t, J=5.3 Hz, 2H), 3.45 (dd, J=11.1, 5.2 Hz,2H).

Step 11:3-{4-[(2-Aminoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-(3-bromo-4-fluorophenyl)-1,2,4-oxadiazol-5(4H)-onehydrochloride

Sodium iodide (1080 g, 7.2 mol) was added to3-{4-[(2-azidoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-(3-bromo-4-fluorophenyl)-1,2,4-oxadiazol-5(4H)-one(500 g, 1.22 mol) in methanol (6 L). The mixture was allowed to stir for30 min during which time a mild exotherm was observed.Chlorotrimethylsilane (930 mL, 7.33 mol) was added as a solution inmethanol (1 L) dropwise at a rate so that the temperature did not exceed35° C., and the reaction was allowed to stir for 3.5 h at ambienttemperature. The reaction was neutralized with 33 wt % solution ofsodium thiosulfate pentahydrate in water (˜1.5 L), diluted with water (4L), and the pH adjusted to 9 carefully with solid potassium carbonate(250 g—added in small portions: watch foaming). Di-tert-butyldicarbonate (318 g, 1.45 mol) was added and the reaction was allowed tostir at room temperature. Additional potassium carbonate (200 g) wasadded in 50 g portions over 4 h to ensure that the pH was still at orabove 9. After stirring at room temperature overnight, the solid wasfiltered, triturated with water (2 L), and then MTBE (1.5 L). A total of11 runs were performed (5.5 kg, 13.38 mol). The combined solids weretriturated with 1:1 THF:dichloromethane (24 L, 4 runs in a 20 L rotaryevaporator flask, 50° C., 1 h), filtered, and washed withdichloromethane (3 L each run) to afford an off-white solid. The crudematerial was dissolved at 55° C. tetrahydrofuran (5 mL/g), treated withdecolorizing carbon (2 wt %) and silica gel (2 wt %), and filtered hotthrough celite to afford the product as an off-white solid (5122 g). Thecombined MTBE, THF, and dichloromethane filtrates were concentrated invacuo and chromatographed (2 kg silica gel, heptane with a 0-100% ethylacetate gradient, 30 L) to afford more product (262 g). The combinedsolids were dried to a constant weight in a convection oven (5385 g,83%).

In a 22 L flask was charged hydrogen chloride (4 N solution in1,4-dioxane, 4 L, 16 mol). tert-Butyl[2-({4-[4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}amino)ethyl]carbamate(2315 g, 4.77 mol) was added as a solid in portions over 10 min. Theslurry was stirred at room temperature and gradually became a thickpaste that could not be stirred. After sitting overnight at roomtemperature, the paste was slurried in ethyl acetate (10 L), filtered,re-slurried in ethyl acetate (5 L), filtered, and dried to a constantweight to afford the desired product as a white solid (combined withother runs, 5 kg starting material charged, 4113 g, 95%). LCMS forC₁₂H₁₁BrFN₆O₃ (M+H)⁺: m/z=384.9, 386.9. ¹H NMR (400 MHz, DMSO-d₆): δ8.12 (m, 4H), 7.76 (m, 1H), 7.58 (t, J=8.7 Hz, 1H), 6.78 (t, J=6.1 Hz,1H), 3.51 (dd, J=11.8, 6.1 Hz, 2H), 3.02 (m, 2H).

Step 12: tert-Butyl({[2-({4-[4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}amino)ethyl]amino}sulfonyl)carbamate

A 5 L round bottom flask was charged with chlorosulfonyl isocyanate[Aldrich, product #142662] (149 mL, 1.72 mol) and dichloromethane (1.5L) and cooled using an ice bath to 2° C. tert-Butanol (162 mL, 1.73 mol)in dichloromethane (200 mL) was added dropwise at a rate so that thetemperature did not exceed 10° C. The resulting solution was stirred atroom temperature for 30-60 min to provide tert-butyl[chlorosulfonyl]carbamate.

A 22 L flask was charged with3-{4-[(2-aminoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-(3-bromo-4-fluorophenyl)-1,2,4-oxadiazol-5(4H)-onehydrochloride (661 g, 1.57 mol) and 8.5 L dichloromethane. After coolingto −15° C. with an ice/salt bath, the solution of tert-butyl[chlorosulfonyl]carbamate (prepared as above) was added at a rate sothat the temperature did not exceed −10° C. (addition time 7 min). Afterstirring for 10 min, triethylamine (1085 mL, 7.78 mol) was added at arate so that the temperature did not exceed −5° C. (addition time 10min). The cold bath was removed, the reaction was allowed to warm to 10°C., split into two portions, and neutralized with 10% conc HCl (4.5 Leach portion). Each portion was transferred to a 50 L separatory funneland diluted with ethyl acetate to completely dissolve the white solid(˜25 L). The layers were separated, and the organic layer was washedwith water (5 L), brine (5 L), and the solvents removed in vacuo toafford an off-white solid. The solid was triturated with MTBE (2×1.5 L)and dried to a constant weight to afford a white solid. A total of 4113g starting material was processed in this manner (5409 g, 98%). ¹H NMR(400 MHz, DMSO-d₆): δ 10.90 (s, 1H), 8.08 (dd, J=6.2, 2.5 Hz, 1H), 7.72(m, 1H), 7.59 (t, J=8.6 Hz, 1H), 6.58 (t, J=5.7 Hz, 1H), 3.38 (dd,J=12.7, 6.2 Hz, 2H), 3.10 (dd, J=12.1, 5.9 Hz, 2H), 1.41 (s, 9H).

Step 13:N-[2-({4-[4-(3-Bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}amino)ethyl]sulfamide

To a 22 L flask containing 98:2 trifluoroacetic acid:water (8.9 L) wasadded tert-butyl({[2-({4-[4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}amino)ethyl]amino}sulfonyl)carbamate(1931 g, 3.42 mol) in portions over 10 minutes. The resulting mixturewas stirred at room temperature for 1.5 h, the solvents removed invacuo, and chased with dichloromethane (2 L). The resulting solid wastreated a second time with fresh 98:2 trifluoroacetic acid:water (8.9L), heated for 1 h at 40-50° C., the solvents removed in vacuo, andchased with dichloromethane (3×2 L). The resulting white solid was driedin a vacuum drying oven at 50° C. overnight. A total of 5409 g wasprocessed in this manner (4990 g, quant. yield). LCMS for C₁₂H₁₂BrFN₇O₅S(M+H)⁺: m/z=463.9, 465.9. ¹H NMR (400 MHz, DMSO-d₆): δ 8.08 (dd, J=6.2,2.5 Hz, 1H), 7.72 (m, 1H), 7.59 (t, J=8.7 Hz, 1H), 6.67 (t, J=5.9 Hz,1H), 6.52 (t, J=6.0 Hz, 1H), 3.38 (dd, J=12.7, 6.3 Hz, 2H), 3.11 (dd,J=12.3, 6.3 Hz).

Step 14:4-({2-[(Aminosulfonyl)amino]ethyl}amino)-N-(3-bromo-4-fluorophenyl)-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide

To a crude mixture ofN-[2-({4-[4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}amino)ethyl]sulfamide(2.4 mol) containing residual amounts of trifluoroacetic acid stirringin a 22 L flask was added THF (5 L). The resulting solution was cooledto 0° C. using an ice bath and 2 N NaOH (4 L) was added at a rate sothat the temperature did not exceed 10° C. After stirring at ambienttemperature for 3 h (LCMS indicated no starting material remained), thepH was adjusted to 3-4 with concentrated HCl (˜500 mL). The THF wasremoved in vacuo, and the resulting mixture was extracted with ethylacetate (15 L). The organic layer was washed with water (5 L), brine (5L), and the solvents removed in vacuo to afford a solid. The solid wastriturated with MTBE (2×2 L), combined with three other reactions of thesame size, and dried overnight in a convection oven to afford a whitesolid (3535 g). The solid was recrystallized (3×22 L flasks, 2:1water:ethanol, 14.1 L each flask) and dried in a 50° C. convection ovento a constant weight to furnish the title compound as an off-white solid(3290 g, 78%). LCMS for C₁₁H₁₄BrFN₇O₄S (M+H)⁺: m/z=437.9, 439.9. ¹H NMR(400 MHz, DMSO-d₆): δ 11.51 (s, 1H), 8.90 (s, 1H), 7.17 (t, J=8.8 Hz,1H), 7.11 (dd, J=6.1, 2.7 Hz, 1H), 6.76 (m, 1H), 6.71 (t, J=6.0 Hz, 1H),6.59 (s, 2H), 6.23 (t, J=6.1 Hz, 1H), 3.35 (dd, J=10.9, 7.0 Hz, 2H),3.10 (dd, J=12.1, 6.2 Hz, 2H).

Example 2N-(3-Bromo-4-fluorophenyl)-N′-hydroxy-4-({2-[(methylsulfonyl)amino]ethyl}amino)-1,2,5-oxadiazole-3-carboximidamide

The title compound was prepared according to the procedure of Example 21step E, usingN-hydroxy-4-({2-[(methylsulfonyl)amino]ethyl}amino)-1,2,5-oxadiazole-3-carboximidamideand 3-bromo-4-fluoroaniline [Oakwood Products, Inc., product #013091] asthe starting materials. LCMS for C₁₂H₁₅BrFN₆O₄S (M+H)⁺: m/z=437.0,439.0. ¹H NMR (400 MHz, DMSO-d₆): δ 11.49 (s, 1H), 8.90 (s, 1H), 7.17(m, 2H), 7.09 (dd, J=6.3, 2.5 Hz, 1H), 6.26 (t, J=6.1 Hz, 1H), 3.33 (m,2H), 3.13 (q, J=6.0 Hz, 2H), 2.89 (s, 3H).

Example 34-({3-[(Aminosulfonyl)amino]propyl}amino)-N-(3-bromo-4-fluorophenyl)-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide

Step 1:3-(4-Amino-1,2,5-oxadiazol-3-yl)-4-(3-bromo-4-fluorophenyl)-1,2,4-oxadiazol-5(4H)-one

The desired compound was prepared according to the procedure of Example9, step 1, using4-amino-N-(3-bromo-4-fluorophenyl)-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide[see U. S. Pat. App. Pub. No. 2006/0258719] as the starting material in98% yield. LCMS for C₁₀H₆BrFN₅O₃ (M+H)⁺: m/z=342.0, 344.0. ¹H NMR (400MHz, DMSO-d₆): δ 8.06 (dd, J=6.2, 2.5 Hz, 1H), 7.72-7.67 (m, 1H), 7.58(dd, J=8.7, 8.7 Hz, 1H), 6.60 (s, 2H).

Step 2:N-{4-[4-(3-Bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}-2,2,2-trifluoroacetamide

The desired compound was prepared according to the procedure of Example9, step 2, using3-(4-amino-1,2,5-oxadiazol-3-yl)-4-(3-bromo-4-fluorophenyl)-1,2,4-oxadiazol-5(4H)-oneas the starting material in 81% yield. LCMS for C₁₂H₅BrF₄N₅O₄ (M+H)⁺:m/z=437.9, 439.9. ¹H NMR (400 MHz, DMSO-d₆): δ 7.92-7.89 (m, 1H),7.54-7.52 (m, 2H).

Step 3:4-(3-Bromo-4-fluorophenyl)-3-{4-[(3-methoxypropyl)amino]-1,2,5-oxadiazol-3-yl}-1,2,4-oxadiazol-5(4H)-one

A solution of 3-methoxypropan-1-ol [Fluka product #38457] (3.1 mL, 32mmol) and triphenylphosphine (8.4 g, 32 mmol) in tetrahydrofuran (93 mL)at 0° C. was treated with diisopropyl azodicarboxylate (6.7 mL, 34 mmol)dropwise. The reaction mixture was stirred at 0° C. for 15 min, treatedwith a solution ofN-{4-[4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}-2,2,2-trifluoroacetamide(10 g, 23 mmol) in tetrahydrofuran (47 mL), and stirred at 25° C. for 72h. The reaction mixture was concentrated, diluted with ethyl acetate(200 mL), treated with trifluoroacetic acid (20 mL) and water (20 mL),and heated at 50° C. for 6 h. The reaction mixture was concentrated,rediluted with ethyl acetate (200 mL) and washed with water (3×80 mL),saturated sodium bicarbonate (2×80 mL) and brine (80 mL), dried overanhydrous sodium sulfate, filtered, and concentrated to a crude residue.This material was purified on silica gel to give the desired product(6.4 g, 54%) as a white solid. LCMS for C₁₄H₁₄BrFN₅O₄ (M+H)⁺: m/z=414.0,416.0.

Step 4:4-(3-Bromo-4-fluorophenyl)-3-{4-[(3-hydroxypropyl)amino]-1,2,5-oxadiazol-3-yl}-1,2,4-oxadiazol-5(4H)-one

A solution of4-(3-bromo-4-fluorophenyl)-3-{4-[(3-methoxypropyl)amino]-1,2,5-oxadiazol-3-yl}-1,2,4-oxadiazol-5(4H)-one(6.3 g, 14 mmol) in dichloromethane (60 mL) at −78° C. was treated with1 M boron tribromide in dichloromethane (28 mL, 28 mmol) and stirred at25° C. for 2 h. The reaction mixture was cooled to 0° C. and quenchedwith saturated sodium bicarbonate (100 mL). The aqueous layer wasseparated and extracted with dichloromethane (2×150 mL). The combinedorganic layers were washed with brine (100 mL), dried over anhydroussodium sulfate, filtered, and concentrated to a crude off-white solid.This material was purified on silica gel to give the desired product(4.0 g, 73%) as a white solid. LCMS for C₁₃H₁₂BrFN₅O₄ (M+H)⁺: m/z=400.0,402.0. ¹H NMR (400 MHz, DMSO-d₆): δ 8.07 (dd, J=6.2, 2.5 Hz, 1H),7.72-7.68 (m, 1H), 7.59 (dd, J=8.8, 8.6 Hz, 1H), 6.54 (t, J=5.7 Hz, 1H),4.60 (t, J=5.1 Hz, 1H), 3.48-3.43 (m, 2H), 3.32-3.26 (m, 2H), 1.74-1.67(m, 2H).

Step 5:3-{4-[(3-Azidopropyl)amino]-1,2,5-oxadiazol-3-yl}-4-(3-bromo-4-fluorophenyl)-1,2,4-oxadiazol-5(4H)-one

A solution of4-(3-bromo-4-fluorophenyl)-3-{4-[(3-hydroxypropyl)amino]-1,2,5-oxadiazol-3-yl}-1,2,4-oxadiazol-5(4H)-one(3.0 g, 7.5 mmol) in dichloromethane (27 mL) was treated withmethanesulfonyl chloride (0.75 mL, 9.7 mmol) andN,N-diisopropylethylamine (2.6 mL, 15 mmol) and stirred at 25° C. for 2h. The reaction mixture was diluted with water (20 mL) and extractedwith dichloromethane (20 mL). The organic layer was separated, driedover anhydrous sodium sulfate, filtered, and concentrated to give themesylate which was used without further purification. A solution of thecrude mesylate in N,N-dimethylformamide (24 mL) was treated with sodiumazide (0.73 g, 11 mmol) and heated at 85° C. for 2 h. The reactionmixture was diluted with ethyl acetate (300 mL) and washed with water(100 mL), saturated sodium bicarbonate (100 mL), and brine (100 mL),dried over anhydrous sodium sulfate, filtered, and concentrated to givethe desired product (3.2 g, 99%). This material was used without furtherpurification. LCMS for C₁₃H₁₀BrFN₈O₃Na (M+Na)±: m/z=446.9, 448.9.

Step 6:3-{4-[(3-Aminopropyl)amino]-1,2,5-oxadiazol-3-yl}-4-(3-bromo-4-fluorophenyl)-1,2,4-oxadiazol-5(4H)-onehydroiodide

A solution of3-{4-[(3-azidopropyl)amino]-1,2,5-oxadiazol-3-yl}-4-(3-bromo-4-fluorophenyl)-1,2,4-oxadiazol-5(4H)-one(2.0 g, 4.7 mmol) in methanol (36 mL) was treated with sodium iodide(4.2 g, 28 mmol) and stirred at 25° C. for 5 min. The reaction mixturewas treated with a solution of chlorotrimethylsilane (3.6 mL, 28 mmol)in methanol (7 mL) dropwise and stirred at 25° C. for 40 min. Thereaction mixture was slowly poured into a solution of sodium thiosulfate(5.0 g, 32 mmol) in water (200 mL) that was cooled at 0° C. The solidthat precipitated was filtered, washed with water, and dried to give thedesired product (2.3 g, 93%) as a solid. LCMS for C₁₃H₁₃BrFN₆O₃ (M+H)⁺:m/z=399.0, 401.0. ¹H NMR (400 MHz, DMSO-d₆): δ 8.08 (dd, J=6.1, 2.3 Hz,1H), 7.74-7.70 (m, 1H), 7.60 (dd, J=8.8, 8.6 Hz, 1H), 7.22 (br s, 2H),6.69 (br s, 1H), 2.81-2.77 (m, 2H), 1.86-1.79 (m, 2H).

Step 7:N-[3-({4-[4-(3-Bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}amino)propyl]sulfamide

A solution of3-{4-[(3-aminopropyl)amino]-1,2,5-oxadiazol-3-yl}-4-(3-bromo-4-fluorophenyl)-1,2,4-oxadiazol-5(4H)-onehydroiodide (150 mg, 0.28 mmol) and sulfamide (160 mg, 1.7 mmol) inpyridine (2.5 mL) was heated in a microwave at 130° C. for 10 min. Thereaction mixture was concentrated to give a crude residue. This materialwas purified by preparative LCMS to give the desired product (96 mg,71%) as a solid. LCMS for C₁₃H₁₄BrFN₇O₅S (M+H)⁺: m/z=478.0, 480.0. ¹HNMR (400 MHz, DMSO-d₆): δ 8.07 (dd, J=6.2, 2.5 Hz, 1H), 7.73-7.69 (m,1H), 7.59 (dd, J=8.8, 8.6 Hz, 1H), 6.57-6.51 (m, 4H), 3.31-3.26 (m, 2H),2.92-2.87 (m, 2H), 1.79-1.72 (m, 2H).

Step 8:4-({3-[(Aminosulfonyl)amino]propyl}amino)-N-(3-bromo-4-fluorophenyl)-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide

A solution ofN-[3-({4-[4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}amino)propyl]sulfamide(35 mg, 73 μmol) in methanol (1 mL) was treated with 2 M NaOH (0.3 mL,0.6 mmol) and stirred at 25° C. for 30 min. The reaction mixture wastreated with acetic acid (50 μL, 0.9 mmol), filtered, and purified bypreparative LCMS to give the desired product (14 mg, 42%) as a solid.LCMS for C₁₂H₁₆BrFN₇O₄S (M+H)⁺: m/z=451.8, 453.9. ¹H NMR (400 MHz,DMSO-d₆): δ 11.5 (s, 1H), 8.89 (s, 1H), 7.17 (dd, J=8.8, 8.6 Hz, 1H),7.09 (dd, J=6.1, 2.7 Hz, 1H), 6.76-6.72 (m, 1H), 6.56 (dd, J=6.1, 6.1Hz, 1H), 6.51 (s, 2H), 6.17 (dd, J=5.9, 5.9 Hz, 1H), 3.27-3.21 (m, 2H),2.94-2.88 (m, 2H), 1.78-1.71 (m, 2H).

Example 4N-(3-Bromo-4-fluorophenyl)-N′-hydroxy-4-({3-[(methylsulfonyl)amino]propyl}amino)-1,2,5-oxadiazole-3-carboximidamide

Step 1: tert-Butyl {3-[(methylsulfonyl)amino]propyl}carbamate

The desired compound was prepared according to the procedure of Example21, step 1, using N-(3-aminopropyl)(tert-butoxy)carboxamide [Aldrichproduct #436992] as the starting material in 70% yield. LCMS forC₄H₁₃N₂O₂S ([M-Boc+H]+H)⁺: m/z=153.1.

Step 2: N-(3-Aminopropyl)methanesulfonamide hydrochloride

The desired compound was prepared according to the procedure of Example21, step 2, using tert-butyl {3-[(methylsulfonyl)amino]propyl}carbamateas the starting material. LCMS for C₄H₁₃N₂O₂S (M+H)⁺: m/z=153.1.

Step 3:4-Amino-N′-hydroxy-N-{3-[(methylsulfonyl)amino]propyl}-1,2,5-oxadiazole-3-carboximidamide

The desired compound was prepared according to the procedure of Example21, step 3, using N-(3-aminopropyl)methanesulfonamide hydrochloride and4-amino-N-hydroxy-1,2,5-oxadiazole-3-carboximidoyl chloride [madeaccording to Example 9, steps 1 through 2] as the starting materials in19% yield.

Step 4:1V-Hydroxy-4-({3-[(methylsulfonyl)amino]propyl}amino)-1,2,5-oxadiazole-3-carboximidamide

The desired compound was prepared according to the procedure of Example21, step 4, using 4-amino-N′-hydroxy-N-{3-[(methylsulfonyl)amino]propyl}-1,2,5-oxadiazole-3-carboximidamide as thestarting material. LCMS for C₇H₁₅N₆O₄S (M+H)⁺: m/z=279.0.

Step 5:N-(3-Bromo-4-fluorophenyl)-N′-hydroxy-4-({3-[(methylsulfonyl)amino]propyl}amino)-1,2,5-oxadiazole-3-carboximidamide

The title compound was prepared according to the procedure of Example21, step 5, usingN′-hydroxy-4-({3-[(methylsulfonyl)amino]propyl}amino)-1,2,5-oxadiazole-3-carboximidamideand 3-bromo-4-fluoroaniline [Oakwood Products, Inc., product #013091] asthe starting materials in 12% yield. LCMS for C₁₃H₁₇BrFN₆O₄S (M+H)⁺:m/z=451.0, 453.0. ¹H NMR (400 MHz, CD3OD): δ 7.12 (dd, J=5.9, 2.4 Hz,1H), 7.05 (t, J=8.7 Hz, 1H), 6.83 (m, 1H), 3.39 (t, J=6.8 Hz, 2H), 3.14(t, J=6.6 Hz, 2H), 2.94 (s, 3H), 1.87 (m, 2H).

Example 54-({2-[(Aminosulfonyl)amino]ethyl}amino)-N-(3-chloro-4-fluorophenyl)-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide

Step 1:3-(4-Amino-1,2,5-oxadiazol-3-yl)-4-(3-chloro-4-fluorophenyl)-1,2,4-oxadiazol-5(4H)-one

A solution of4-amino-N-(3-chloro-4-fluorophenyl)-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide(80 g, 0.29 mol) [see US Pat. App. Pub. No. 2006/0258719] intetrahydrofuran (500 mL) was treated with a solution of1,1′-carbonyldiimidazole (53 g, 0.32 mol) in tetrahydrofuran (200 mL)and heated at reflux for 1 h. The reaction mixture was cooled to 25° C.and concentrated to the point where a large amount of solidprecipitated. The heterogeneous mixture was diluted with ethyl acetate(1.5 L) and washed with 1 N HCl (2×300 mL), water (300 mL), and brine(200 mL). The organic layer was separated, dried over anhydrous sodiumsulfate, filtered, and concentrated to give the desired product (88 g,quantitative) as an off-white solid. This material was used withoutfurther purification. LCMS for C₁₀H₆ClFN₅O₃ (M+H)⁺: m/z=298.0. ¹H NMR(400 MHz, DMSO-d₆): δ 7.96 (dd, J=6.6, 2.3 Hz, 1H), 7.69-7.60 (m, 2H),6.60 (s, 2H).

Step 2:N-{4-[4-(3-Chloro-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}-2,2,2-trifluoroacetamide

A solution of3-(4-amino-1,2,5-oxadiazol-3-yl)-4-(3-chloro-4-fluorophenyl)-1,2,4-oxadiazol-5(4H)-one(15 g, 50 mmol) in dichloromethane (120 mL) was treated withtrifluoroacetic anhydride (14 mL, 100 mmol), cooled to 0° C., andtreated with pyridine (8.2 mL, 100 mmol). The reaction mixture wasstirred at 25° C. for 10 min, cooled to 0° C., and quenched with water(10 mL). The reaction mixture was diluted with ethyl acetate (500 mL)and washed with 1 N HCl (300 mL), water (2×200 mL), and brine (200 mL).The organic layer was separated, dried over anhydrous sodium sulfate,filtered, and concentrated to 50 mL volume. This solution was warmed(˜40-50° C.) and treated with hexanes (600 mL) under vigorous stirring,followed by petroleum ether (200 mL). The mixture was stirred at 0° C.for 30 min and the solid was collected by filtration, washed withhexanes, and dried to give the desired product (19.7 g, 99%) as a whitesolid. LCMS for C₁₂H₅ClF₄N₅O₄ (M+H)⁺: m/z=394.0. ¹H NMR (400 MHz,DMSO-d₆): δ 7.82 (dd, J=6.6, 2.5 Hz, 1H), 7.59 (dd, J=9.0, 9.0 Hz, 1H),7.52-7.47 (m, 1H).

Step 3:4-(3-Chloro-4-fluorophenyl)-3-(4-{[2-(tritylamino)ethyl]amino}-1,2,5-oxadiazol-3-yl)-1,2,4-oxadiazol-5(4H)-one

A solution of 2-(tritylamino)ethanol (10 g, 33 mmol) [EP599220 and J.Org. Chem. (2001), 66, 7615] and triphenylphosphine (8.7 g, 33 mmol) intetrahydrofuran (65 mL) at 0° C. was treated with diisopropylazodicarboxylate (7.0 mL, 35 mmol) dropwise. The reaction mixture wasstirred at 0° C. for 15 min, treated with a solution ofN-{4-[4-(3-chloro-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}-2,2,2-trifluoroacetamide(9.3 g, 24 mmol) in tetrahydrofuran (28 mL), and stirred at 25° C. for16 h. The reaction mixture was concentrated, diluted with ethyl acetate(350 mL), cooled to 0° C., treated with 1 N HCl (200 mL), and stirred at25° C. for 1 h. The reaction mixture was treated with additional 1 N HCl(150 mL) and stirred at 25° C. for 3 h. The organic layer was separated,washed with saturated sodium bicarbonate (200 mL) and brine (100 mL),dried over anhydrous sodium sulfate, filtered, and concentrated to ayellow foam which was reconcentrated from hexanes to give an oily solid.The oily solid was treated with methyl tert-butyl ether (50 mL) andstirred to give a heterogeneous mixture. The solid was filtered, washedwith methyl tert-butyl ether (30 mL), and dried to give the desiredproduct (10 g, 74%) as a white solid. LCMS for C₃₁H₂₄ClFN₆O₃Na (M+Na)±:m/z=605.2. ¹H NMR (300 MHz, DMSO-d₆): δ 7.97 (dd, J=6.7, 2.6 Hz, 1H),7.71-7.66 (m, 1H), 7.60 (dd, J=9.1, 8.8 Hz, 1H), 7.40-7.37 (m, 6H),7.28-7.23 (m, 6H), 7.18-7.12 (m, 3H), 6.59 (dd, J=5.9, 5.6 Hz, 1H),3.37-3.31 (m, 2H), 2.96 (dd, J=7.6, 7.6 Hz, 1H), 2.27-2.19 (m, 2H).

Step 4:3-{4-[(2-Aminoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-(3-chloro-4-fluorophenyl)-1,2,4-oxadiazol-5(4H)-onehydrochloride

A premixed solution of triisopropylsilane (3.4 mL, 17 mmol) andtrifluoroacetic acid (44 mL, 570 mmol) was added to4-(3-chloro-4-fluorophenyl)-3-(4-{[2-(tritylamino)ethyl]amino}-1,2,5-oxadiazol-3-yl)-1,2,4-oxadiazol-5(4H)-one(6.5 g, 11 mmol) and the resulting suspension was stirred at 25° C. for30 min. The reaction mixture was filtered and washed withtrifluoroacetic acid. The filtrate was concentrated to an oil which wasdiluted with methanol (25 mL), cooled to 0° C., treated with 4 M HCl in1,4-dioxane (14 mL), and stirred at 25° C. for 15 min. The mixture wasconcentrated to a solid that was treated with diethyl ether (50 mL) andfiltered. The solid was washed with diethyl ether (50 mL) and dried togive the desired product (4.1 g, 98%) as a white solid. LCMS forC₁₂H₁₁ClFN₆O₃ (M+H)⁺: m/z=341.1. ¹H NMR (300 MHz, DMSO-d₆): δ 8.05-8.00(m, 4H), 7.75-7.69 (m, 1H), 7.64 (dd, J=9.1, 8.8 Hz, 1H), 6.77 (dd,J=5.9, 5.9 Hz, 1H), 3.54-3.47 (m, 2H), 3.04-2.99 (m, 2H).

Step 5:N-[2-({4-[4-(3-Chloro-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}amino)ethyl]sulfamide

A solution of chlorosulfonyl isocyanate (2.0 mL, 23 mmol) indichloromethane (70 mL) was treated with t-butyl alcohol (2.2 mL, 23mmol) at 0° C. and stirred at 25° C. for 1 h. This mixture was added toa suspension of3-{4-[(2-aminoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-(3-chloro-4-fluorophenyl)-1,2,4-oxadiazol-5(4H)-onehydrochloride (4.3 g, 11 mmol) in dichloromethane (70 mL). The reactionmixture was treated with a solution of triethylamine (6.3 mL, 45 mmol)in dichloromethane (20 mL) at 0° C. and stirred at 25° C. for 3 h. Thereaction mixture was diluted with 0.1 N HCl and extracted with ethylacetate (2×100 mL). The combined organic layers were washed with brine(100 mL), dried over anhydrous sodium sulfate, filtered, andconcentrated to a white solid. The white solid was diluted withdichloromethane (100 mL), treated with trifluoroacetic acid (20 mL), andstirred at 25° C. for 3 h. The reaction mixture was concentrated to acrude residue that was purified by silica gel chromatography to give thedesired product (3.7 g, 78%) as a white solid. LCMS for C₁₂H₁₂ClFN₇O₅S(M+H)⁺: m/z=420.0. ¹H NMR (300 MHz, DMSO-d₆): δ 7.98 (dd, J=6.4, 2.1 Hz,1H), 7.70-7.60 (m, 2H), 6.66 (t, J=5.9 Hz, 1H), 6.57 (s, 2H), 6.52 (t,J=5.9 Hz, 1H), 3.42-3.35 (m, 2H), 3.13-3.06 (m, 2H).

Step 6:4-({2-[(Aminosulfonyl)amino]ethyl}amino)-N-(3-chloro-4-fluorophenyl)-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide

A solution ofN-[2-({4-[4-(3-chloro-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}amino)ethyl]sulfamide(3.7 g, 8.8 mmol) in methanol (70 mL) was treated with 2 M NaOH (18 mL,35 mmol) and stirred at 25° C. for 2 h. The reaction mixture wasquenched with 6 N HCl to pH-7 and the methanol was removed under reducedpressure. The solid that precipitated was filtered and washed with waterto give the desired product (3.2 g, 92%) as a white solid. LCMS forC₁₁H₁₄ClFN₇O₄S (M+H)⁺: m/z=394.0. ¹H NMR (400 MHz, DMSO-d₆): δ 7.96 (dd,J=6.8, 2.1 Hz, 0.05H), 7.32-7.29 (m, 0.1H), 7.18 (dd, J=9.1, 9.1 Hz,0.95H), 6.93 (dd, J=6.4, 2.7 Hz, 0.95H), 6.71-6.66 (m, 0.95H), 6.33 (brs, 1H), 3.35-3.27 (m, 2H), 3.10-3.06 (m, 2H).

Example 6N-(3-Chloro-4-fluorophenyl)-N′-hydroxy-4-({2-[(methylsulfonyl)amino]ethyl}amino)-1,2,5-oxadiazole-3-carboximidamide

The title compound was prepared according to the procedure of Example 21step E, using N′-hydroxy-4-({2-[(methylsulfonyl)amino]ethyl}amino)-1,2,5-oxadiazole-3-carboximidamide and3-chloro-4-fluoroaniline [Aldrich, product #228583] as the startingmaterials. LCMS for C₁₂H₁₅ClFN₆O₄S (M+H)⁺: m/z=393.0. ¹H NMR (400 MHz,DMSO-d₆): δ 11.50 (s, 1H), 8.91 (s, 1H), 7.19 (m, 2H), 6.96 (dd, J=6.7,2.5 Hz, 1H), 6.71 (m, 1H), 6.26 (t, J=6.4 Hz, 1H), 3.32 (m, 2H), 3.13(q, J=5.8 Hz, 2H), 2.89 (s, 3H).

Example 74-({3-[(Aminosulfonyl)amino]propyl}amino)-N-(3-chloro-4-fluorophenyl)-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide

Step 1: 3-[(Diphenylmethylene)amino]propan-1-ol

A solution of 3-amino-1-propanol [Aldrich product #A76400] (2.0 mL, 26mmol) in dichloromethane (79 mL) was treated with benzophenone imine(4.4 mL, 26 mmol) and stirred at 25° C. for 16 h. The reaction mixturewas filtered and the filtrate was concentrated to give the desiredproduct (6.3 g, quantitative) as an oil. This material was used withoutfurther purification. LCMS for C₁₆H₁₈NO (M+H)⁺: m/z=240.2.

Step 2:3-{4-[(3-Aminopropyl)amino]-1,2,5-oxadiazol-3-yl}-4-(3-chloro-4-fluorophenyl)-1,2,4-oxadiazol-5(4H)-onetrifluoroacetate

A solution of 3-[(diphenylmethylene)amino]propan-1-ol (80 mg, 0.33 mmol)and triphenylphosphine (93 mg, 0.36 mmol) in tetrahydrofuran (1 mL) at0° C. was treated with diisopropyl azodicarboxylate (75 μL, 0.38 mmol)dropwise. The reaction mixture was stirred at 0° C. for 15 min, treatedwith a solution ofN-{4-[4-(3-chloro-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}-2,2,2-trifluoroacetamide(100 mg, 0.25 mmol) in tetrahydrofuran (0.5 mL), and stirred at 25° C.for 16 h. The reaction mixture was treated with trifluoroacetic acid (1mL), stirred at 25° C. for 3 h, and concentrated to a crude residue.This material was purified by preparative LCMS to give the desiredproduct (18 mg, 15%). LCMS for C₁₃H₁₃ClFN₆O₃ (M+H)⁺: m/z=355.1

Step 3:4-({3-[(Aminosulfonyl)amino]propyl}amino)-N-(3-chloro-4-fluorophenyl)-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide

The desired compound was prepared according to the procedure of Example19, step 7, using3-{4-[(3-aminopropyl)amino]-1,2,5-oxadiazol-3-yl}-4-(3-chloro-4-fluorophenyl)-1,2,4-oxadiazol-5(4H)-onetrifluoroacetate as the starting material in 34% yield. LCMS forC₁₂H₁₆ClFN₇O₄S (M+H)⁺: m/z=408.1. ¹H NMR (400 MHz, DMSO-d₆): δ 8.90 (s,1H), 7.20 (dd, J=9.2, 9.0 Hz, 1H), 6.96 (dd, J=6.4, 2.7 Hz, 1H),6.72-6.69 (m, 1H), 6.55 (t, J=6.0 Hz, 1H), 6.51 (s, 2H), 6.16 (t, J=5.9Hz, 1H), 3.28-3.21 (m, 2H), 2.93-2.87 (m, 2H), 1.76-1.72 (m, 2H).

Example 8N-(3-Chloro-4-fluorophenyl)-N′-hydroxy-4-({3-[(methylsulfonyl)amino]propyl}amino)-1,2,5-oxadiazole-3-carboximidamide

The title compound was prepared according to the procedure of Example 8,step 5, using N′-hydroxy-4-({3-[(methylsulfonyl)amino]propyl}amino)-1,2,5-oxadiazole-3-carboximidamide [madeaccording to Example 8, steps 1 through 4] and 3-chloro-4-fluoroaniline[Aldrich, product #228583] as the starting materials in 10% yield. LCMSfor C₁₃H₁₇ClFN₆O₄S (M+H)⁺: m/z=407.1. ¹H NMR (400 MHz, CD3OD): δ 7.06(t, J=8.9 Hz, 1H), 6.98 (m, 1H), 6.80 (m, 1H), 3.73 (m, 2H), 3.28 (m,2H), 2.94 (s, 3H), 1.28 (m, 2H).

Example 94-({2-[(Aminosulfonyl)amino]ethyl}amino)-N-[4-fluoro-3-(trifluoromethyl)phenyl]-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide

Step 1:N-[4-Fluoro-3-(trifluoromethyl)phenyl]-N′-hydroxy-4-[(2-methoxyethyl)amino]-1,2,5-oxadiazole-3-carboximidamide

The desired compound was prepared according to the procedure of Example17, step 1, usingN-hydroxy-4-[(2-methoxyethyl)amino]-1,2,5-oxadiazole-3-carboximidoylchloride [made according to Example 5, steps 1 through 5] and3-trifluoromethyl-4-fluoroaniline [Aldrich, product #217778] as thestarting materials in quantitative yield. LCMS for C₁₃H₁₄F₄N₅O₃ (M+H)⁺:m/z=364.0. ¹H NMR (400 MHz, CD3OD): δ 7.15 (m, 2H), 7.08 (m, 1H), 3.60(t, J=5.3 Hz, 2H), 3.46 (t, J=5.3 Hz, 2H), 3.38 (s, 3H).

Step 2:4-[4-Fluoro-3-(trifluoromethyl)phenyl]-3-{4-[(2-methoxyethyl)amino]-1,2,5-oxadiazol-3-yl}-1,2,4-oxadiazol-5(4H)-one

The desired compound was prepared according to the procedure of Example17, step 2, usingN-[4-fluoro-3-(trifluoromethyl)phenyl]-N′-hydroxy-4-[(2-methoxyethyl)amino]-1,2,5-oxadiazole-3-carboximidamideas the starting material in 79% yield. LCMS for C₁₄H₁₂F₄N₅O₄ (M+H)⁺:m/z=390.0. ¹H NMR (400 MHz, DMSO-d₆): δ 8.20 (dd, J=6.3, 2.4 Hz, 1H),8.03 (m, 1H), 7.76 (t, J=9.5 Hz, 1H), 6.41 (t, J=5.7 Hz, 1H), 3.49 (t,J=5.5 Hz, 2H), 3.39 (q, J=5.7 Hz, 2H), 3.25 (s, 3H).

Step 3:4-[4-Fluoro-3-(trifluoromethyl)phenyl]-3-{4-[(2-hydroxyethyl)amino]-1,2,5-oxadiazol-3-yl}-1,2,4-oxadiazol-5(4H)-one

The desired compound was prepared according to the procedure of Example17, step 3, using4-[4-fluoro-3-(trifluoromethyl)phenyl]-3-{4-[(2-methoxyethyl)amino]-1,2,5-oxadiazol-3-yl}-1,2,4-oxadiazol-5(4H)-oneas the starting material in 99% yield. LCMS for C₁₃H₁₀F₄N₅O₄ (M+H)⁺:m/z=376.0. ¹H NMR (400 MHz, DMSO-d₆): δ 8.22 (m, 1H), 8.05 (m, 1H), 7.76(t, J=9.9 Hz, 1H), 6.34 (t, J=5.7 Hz, 1H), 4.87 (t, J=5.2 Hz, 1H), 3.56(q, J=5.5 Hz, 2H), 3.29 (q, J=5.7 Hz, 2H).

Step 4:2-[(4-{4-[4-Fluoro-3-(trifluoromethyl)phenyl]-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl}-1,2,5-oxadiazol-3-yl)amino]ethylmethanesulfonate

The desired compound was prepared according to the procedure of Example17, step 4, using4-[4-fluoro-3-(trifluoromethyl)phenyl]-3-{4-[(2-hydroxyethyl)amino]-1,2,5-oxadiazol-3-yl}-1,2,4-oxadiazol-5(4H)-oneas the starting material in 95% yield. LCMS for C₁₄H₁₂F₄N₅O₆S (M+H)⁺:m/z=454.0. ¹H NMR (400 MHz, DMSO-d₆): δ 8.23 (dd, J=6.5, 2.5 Hz, 1H),8.06 (m, 1H), 7.76 (t, J=9.6 Hz, 1H), 6.76 (t, J=5.8 Hz, 1H), 4.37 (t,J=5.4 Hz, 2H), 3.60 (q, J=5.5 Hz, 2H), 3.17 (s, 3H).

Step 5:3-{4-[(2-Azidoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-[4-fluoro-3-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5(4H)-one

The desired compound was prepared according to the procedure of Example17, step 5, using2-[(4-{4-[4-fluoro-3-(trifluoromethyl)phenyl]-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl}-1,2,5-oxadiazol-3-yl)amino]ethylmethanesulfonate as the starting material in 100% yield. LCMS forC₁₃H₉F₄N₆O₃ (M-N₂+H)⁺: m/z=372.8. ¹H NMR (400 MHz, DMSO-d₆): δ 8.22 (dd,J=6.2, 2.4 Hz, 1H), 8.05 (m, 1H), 7.76 (t, J=9.6 Hz, 1H), 6.75 (t, J=5.9Hz, 1H), 3.53 (t, J=5.9 Hz, 2H), 3.45 (q, J=5.6 Hz, 2H).

Step 6:3-{4-[(2-Aminoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-[4-fluoro-3-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5(4H)-onehydroiodide

The desired compound was prepared according to the procedure of Example17, step 6, using3-{4-[(2-azidoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-[4-fluoro-3-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5(4H)-oneas the starting material in 80% yield. LCMS for C₁₃H₁₁F₄N₆O₃ (M+H)⁺:m/z=375.0. ¹H NMR (400 MHz, DMSO-d₆): δ 8.20 (dd, J=6.2, 2.4 Hz, 1H),8.03 (m, 1H), 7.74 (t, J=9.8 Hz, 1H), 7.10 (br s, 0.4H), 6.68 (t, J=5.5Hz, 1H), 3.42 (q, J=5.8 Hz, 2H), 2.95 (t, J=6.5 Hz, 2H).

Step 7:4-({2-[(Aminosulfonyl)amino]ethyl}amino)-N-[4-fluoro-3-(trifluoromethyl)phenyl]-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide

The title compound was prepared according to the procedure of Example17, step 7, using3-{4-[(2-aminoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-[4-fluoro-3-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5(4H)-onehydroiodide as the starting material in 55% yield. LCMS forC₁₂H₁₄F₄N₇O₄S (M+H)⁺: m/z=428.0. ¹H NMR (400 MHz, DMSO-d₆): δ 11.60 (s,1H), 9.06 (s, 1H), 7.30 (t, J=10.1 Hz, 1H), 7.14 (dd, J=6.1, 2.7 Hz,1H), 7.03 (m, 1H), 6.71 (t, J=5.3 Hz, 1H), 6.58 (s, 2H), 6.23 (t, J=6.2Hz, 1H), 3.36 (q, J=6.5 Hz, 2H), 3.08 (m, 2H).

Example 10N-[4-Fluoro-3-(trifluoromethyl)phenyl]-N′-hydroxy-4-({2-[(methylsulfonyl)amino]ethyl}amino)-1,2,5-oxadiazole-3-carboximidamide

The title compound was prepared according to the procedure of Example 21step E, usingN-hydroxy-4-({2-[(methylsulfonyl)amino]ethyl}amino)-1,2,5-oxadiazole-3-carboximidamideand 3-trifluoromethyl-4-fluoroaniline [Aldrich, product #217778] as thestarting materials. LCMS for C₁₃H₁₅F₄N₆O₄S (M+H)⁺: m/z=427.0. ¹H NMR(400 MHz, DMSO-d₆): δ 11.60 (s, 1H), 9.07 (s, 1H), 7.30 (t, J=10.1 Hz,1H), 7.18 (t, J=6.0 Hz, 1H), 7.13 (dd, J=6.0, 2.7 Hz, 1H), 7.03 (m, 1H),6.27 (t, J=6.3 Hz, 1H), 3.32 (m, 2H), 3.13 (q, J=6.0 Hz, 2H), 2.89 (s,3H).

Example 114-({3-[(Aminosulfonyl)amino]propyl}amino)-N-[4-fluoro-3-(trifluoromethyl)phenyl]-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide

Step 1:4-Amino-N′-hydroxy-N-(3-methoxypropyl)-1,2,5-oxadiazole-3-carboximidamide

The desired compound was prepared according to the procedure of Example5, step 3, using 3-methoxy-1-propanamine as the starting material in 93%yield. LCMS for C₇H₁₄N₅O₃ (M+H)⁺: m/z=216.1.

Step 2:1V-Hydroxy-4-[(3-methoxypropyl)amino]-1,2,5-oxadiazole-3-carboximidamide

The desired compound was prepared according to the procedure of Example5, step 4, using4-amino-N′-hydroxy-N-(3-methoxypropyl)-1,2,5-oxadiazole-3-carboximidamideas the starting material in 72% yield. LCMS for C₇H₁₄N₅O₃ (M+H)⁺:m/z=216.1. ¹H NMR (300 MHz, DMSO-d₆): δ 10.4 (s, 1H), 6.21-6.13 (m, 3H),3.37 (t, J=6.1 Hz, 2H), 3.28-3.21 (m, 5H), 1.82-1.74 (m, 2H).

Step 3:N-Hydroxy-4-[(3-methoxypropyl)amino]-1,2,5-oxadiazole-3-carboximidoylchloride

The desired compound was prepared according to the procedure of Example5, step 5, usingN′-Hydroxy-4-[(3-methoxypropyl)amino]-1,2,5-oxadiazole-3-carboximidamideas the starting material in quantitative yield. LCMS for C₇H₁₂ClN₄O₃(M+H)⁺: m/z=235.1.

Step 4:N-[4-Fluoro-3-(trifluoromethyl)phenyl]-N′-hydroxy-4-[(3-methoxypropyl)amino]-1,2,5-oxadiazole-3-carboximidamide

The desired compound was prepared according to the procedure of Example5, step 6, usingN-hydroxy-4-[(3-methoxypropyl)amino]-1,2,5-oxadiazole-3-carboximidoylchloride and 4-fluoro-3-(trifluoromethyl)benzeneamine as the startingmaterials in 87% yield. LCMS for C₁₄H₁₆F₄N₅O₃ (M+H)⁺: m/z=378.1. ¹H NMR(400 MHz, DMSO-d₆): δ 11.5 (s, 1H), 9.05 (s, 1H), 7.30 (dd, J=10.0, 9.6Hz, 1H), 7.13-7.11 (m, 1H), 7.05-7.00 (m, 1H), 6.22 (t, J=5.7 Hz, 1H),3.35-3.32 (m, 2H), 3.25-3.19 (m, 5H), 1.79-1.72 (m, 2H).

Step 5:4-[4-Fluoro-3-(trifluoromethyl)phenyl]-3-{4-[(3-methoxypropyl)amino]-1,2,5-oxadiazol-3-yl}-1,2,4-oxadiazol-5(4H)-one

The desired compound was prepared according to the procedure of Example5, step 7, usingN-[4-fluoro-3-(trifluoromethyl)phenyl]-N′-hydroxy-4-[(3-methoxypropyl)amino]-1,2,5-oxadiazole-3-carboximidamideas the starting material in quantitative yield. LCMS for C₁₅H₁₄F₄N₅O₄(M+H)⁺: m/z=404.0.

Step 6:4-[4-Fluoro-3-(trifluoromethyl)phenyl]-3-{4-[(3-hydroxypropyl)amino]-1,2,5-oxadiazol-3-yl}-1,2,4-oxadiazol-5(4H)-one

The desired compound was prepared according to the procedure of Example7, step 4, using4-[4-fluoro-3-(trifluoromethyl)phenyl]-3-{4-[(3-methoxypropyl)amino]-1,2,5-oxadiazol-3-yl}-1,2,4-oxadiazol-5(4H)-oneas the starting material in 97% yield. LCMS for C₁₄H₁₂F₄N₅O₄ (M+H)⁺:m/z=390.0. ¹H NMR (300 MHz, DMSO-d₆): δ 8.20 (dd, J=6.4, 2.6 Hz, 1H),8.06-8.01 (m, 1H), 7.75 (dd, J=10.0, 9.4 Hz, 1H), 6.53 (t, J=5.7 Hz,1H), 4.59 (t, J=5.0 Hz, 1H), 3.51-3.42 (m, 2H), 3.32-3.26 (m, 2H),1.73-1.68 (m, 2H).

Step 7:3-{4-[(3-Azidopropyl)amino]-1,2,5-oxadiazol-3-yl}-4-[4-fluoro-3-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5(4H)-one

The desired compound was prepared according to the procedure of Example7, step 5, using4-[4-fluoro-3-(trifluoromethyl)phenyl]-3-{4-[(3-hydroxypropyl)amino]-1,2,5-oxadiazol-3-yl}-1,2,4-oxadiazol-5(4H)-oneas the starting material in quantitative yield. LCMS for C₁₄H₁₀F₄N₈O₃Na(M+Na)⁺: m/z=437.0.

Step 8:3-{4-[(3-Aminopropyl)amino]-1,2,5-oxadiazol-3-yl}-4-[4-fluoro-3-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5(4H)-onehydroiodide

The desired compound was prepared according to the procedure of Example7, step 6, using3-{4-[(3-azidopropyl)amino]-1,2,5-oxadiazol-3-yl}-4-[4-fluoro-3-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5(4H)-oneas the starting material in 81% yield. LCMS for C₁₄H₁₃F₄N₆O₃ (M+H)⁺:m/z=389.1. ¹H NMR (300 MHz, DMSO-d₆): δ 8.18 (dd, J=6.4, 2.3 Hz, 1H),8.06-8.01 (m, 1H), 7.72 (dd, J=9.7, 9.4 Hz, 1H), 7.34 (br s, 2H), 6.71(br s, 1H), 2.78-2.73 (m, 2H), 1.85-1.75 (m, 2H).

Step 9:4-({3-[(Aminosulfonyl)amino]propyl}amino)-N-[4-fluoro-3-(trifluoromethyl)phenyl]-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide

The desired compound was prepared according to the procedure of Example19, step 7, using3-{4-[(3-aminopropyl)amino]-1,2,5-oxadiazol-3-yl}-4-[4-fluoro-3-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5(4H)-onehydroiodide as the starting material in 60% yield. LCMS forC₁₃H₁₆F₄N₇O₄S (M+H)⁺: m/z=442.0. ¹H NMR (300 MHz, DMSO-d₆): δ 11.6 (s,1H), 9.08 (s, 1H), 7.31 (dd, J=10.0, 9.4 Hz, 1H), 7.13 (dd, J=6.4, 2.9Hz, 1H), 7.05-6.99 (m, 1H), 6.58 (t, J=6.0 Hz, 1H), 6.52 (s, 2H), 6.17(t, J=5.9 Hz, 1H), 3.28-3.21 (m, 2H), 2.94-2.87 (m, 2H), 1.79-1.72 (m,2H).

Example 12N-[4-Fluoro-3-(trifluoromethyl)phenyl]-N′-hydroxy-4-({3-[(methylsulfonyl)amino]propyl}amino)-1,2,5-oxadiazole-3-carboximidamide

The desired compound was prepared according to the procedure of Example20 using3-{4-[(3-aminopropyl)amino]-1,2,5-oxadiazol-3-yl}-4-[4-fluoro-3-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5(4H)-one hydroiodide as the starting materialin 70% yield. LCMS for C₁₄H₁₇F₄N₆O₄S (M+H)⁺: m/z=441.1. ¹H NMR (400 MHz,DMSO-d₆): δ 11.6 (s, 1H), 9.07 (s, 1H), 7.30 (dd, J=10.0, 9.6 Hz, 1H),7.13 (dd, J=6.2, 2.5 Hz, 1H), 7.05-7.02 (m, 2H), 6.19 (t, J=5.8 Hz, 1H),3.27-3.21 (m, 2H), 2.99-2.94 (m, 2H), 2.87 (s, 3H), 1.76-1.72 (m, 2H).

Example 134-({2-[(Aminosulfonyl)amino]ethyl}amino)-N′-hydroxy-N-[3-(trifluoromethyl)phenyl]-1,2,5-oxadiazole-3-carboximidamide

Step 1:N′-hydroxy-4-[(2-methoxyethyl)amino]-N-[3-(trifluoromethyl)phenyl]-1,2,5-oxadiazole-3-carboximidamide

N-Hydroxy-4-[(2-methoxyethyl)amino]-1,2,5-oxadiazole-3-carboximidoylchloride (1.3 g, 5.0 mmol) [made according to Example 5, steps 1 through5] was stirred in water (10 mL) and warmed to 60° C. for 5 minutes.3-(trifluoromethyl)aniline [Aldrich, product #A41801] (880 mg, 5.5 mmol)was added in one portion and the reaction stirred for 15 minutes. Whileremaining at 60° C., a solution of sodium bicarbonate (630 mg, 7.5 mmol)in water (10 mL) was added dropwise over 5 minutes. The reaction wasstirred at 60° C. for an additional 50 minutes, and then allowed to coolto room temperature. Ethyl acetate (20 mL) and brine (30 mL) were addedto the flask and the organic layer was collected. The aqueous layer wasextracted with ethyl acetate (2×20 mL) and the combined organics weredried over sodium sulfate. The solvent was removed in vacuo to give thedesired product as an orange solid (1.4 g, 80%). LCMS calculated forC₁₃H₁₅F₃N₅O₃ (M+H)⁺: m/z=346.1. ¹H NMR (400 MHz, CD3OD): δ 7.36 (t,J=8.2 Hz, 1H), 7.23 (d, J=7.6 Hz, 1H), 7.09 (s, 1H), 7.02 (d, J=8.2 Hz,1H), 3.60 (t, J=5.2 Hz, 2H), 3.46 (t, J=5.2 Hz, 2H), 3.38 (s, 3H).

Step 2:3-{4-[(2-Methoxyethyl)amino]-1,2,5-oxadiazol-3-yl}-4-[3-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5(4H)-one

N′-Hydroxy-4-[(2-methoxyethyl)amino]-N-[3-(trifluoromethyl)phenyl]-1,2,5-oxadiazole-3-carboximidamide(1.4 g, 3.80 mmol) and 1,1′-carbonyldiimidazole (1.16 g, 7.16 mmol) weredissolved in ethyl acetate (20 mL). The reaction mixture was heated at70° C. for 40 minutes. Additional 1,1′-carbonyldiimidazole (0.26 g, 1.16mmol) was added. After stirring at 70° C. for another 50 minutes, thereaction was allowed to cool to room temperature. Ethyl acetate (20 mL)was added and the crude reaction was washed with 1 N HCl in water (2×20mL). Brine was added to aid in the separation of the first wash. Theorganic layer was dried over sodium sulfate and concentrated in vacuo.Purification by flash chromatography on silica gel with an eluent ofethyl acetate in hexanes gave the desired product (1.3 g, 90%). LCMScalculated for C₁₄H₁₃F₃N₅O₄ (M+H)⁺: m/z=372.0. ¹H NMR (400 MHz,DMSO-d₆): δ 8.07 (s, 1H), 7.92 (m, 2H), 7.79 (t, J=8.1 Hz, 1H), 6.42 (t,J=6.0 Hz, 1H), 3.47 (t, J=5.8 Hz, 2H), 3.38 (q, J=5.0 Hz, 2H), 3.24 (s,3H).

Step 3:3-{4-[(2-Hydroxyethyl)amino]-1,2,5-oxadiazol-3-yl}-4-[3-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5(4H)-one

In a round bottom flask under nitrogen atmosphere,3-{4-[(2-methoxyethyl)amino]-1,2,5-oxadiazol-3-yl}-4-[3-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5(4H)-one(1.3 g, 3.6 mmol) was stirred in dichloromethane (11 mL). Thetemperature was brought to −78° C. and a solution of 1.0 M borontribromide in dichloromethane (7.9 mL, 7.9 mmol) was added dropwise over15 minutes. The reaction was warmed to room temperature over 45 minutesand continued to stir at room temperature for an additional 45 minutes.The reaction was cooled to 0° C. and a saturated solution of sodiumbicarbonate in water (25 mL) was added dropwise over 15 minutes. Afterwarming to room temperature, ethyl acetate (10 mL) and water (10 mL)were added to the flask. The organic layer was collected and the aqueouslayer was extracted with ethyl acetate (2×20 mL). After drying thecombined organic layers over sodium sulfate, the solvent was removed invacuo to give the desired product (1.0 g, 81%). LCMS calculated forC₁₃H₁₁F₃N₅O₄ (M+H)⁺: m/z=358.0. ¹H NMR (400 MHz, DMSO-d₆): δ 8.08 (s,1H), 7.93 (t, J=8.2 Hz, 2H), 7.79 (t, J=8.2 Hz, 1H), 6.35 (t, J=5.7 Hz,1H), 4.86 (br s, 1H), 3.55 (t, J=6.0 Hz, 2H), 3.28 (m, 2H).

Step 4:2-[(4-{5-Oxo-4-[3-(trifluoromethyl)phenyl]-4,5-dihydro-1,2,4-oxadiazol-3-yl}-1,2,5-oxadiazol-3-yl)amino]ethylmethanesulfonate

To a solution of3-{4-[(2-hydroxyethyl)amino]-1,2,5-oxadiazol-3-yl}-4-[3-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5(4H)-one(1.0 g, 2.9 mmol) in ethyl acetate (8.5 mL) was added methanesulfonylchloride (0.29 mL, 3.7 mmol) in one portion. The reaction was stirredfor 5 minutes and triethylamine (0.52 mL, 3.7 mmol) was added, also inone portion. After stirring for an additional 10 minutes, the reactionwas quenched with the addition of water (5 mL). The product wasextracted with ethyl acetate (2×5 mL), dried over sodium sulfate andconcentrated in vacuo to give the desired product (1.2 g, 99%). LCMScalculated for C₁₄H₁₃F₃N₅O₆S (M+H)⁺: m/z=436.0. ¹H NMR (400 MHz,DMSO-d₆): δ 8.10 (s, 1H), 7.92 (m, 2H), 7.80 (t, J=8.2 Hz, 1H), 6.77 (t,J=5.9 Hz, 1H), 4.36 (t, J=5.5 Hz, 2H), 3.58 (m, 2H), 3.17 (s, 3H).

Step 5:3-{4-[(2-Azidoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-[3-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5(4H)-one

2-[(4-{5-Oxo-4-[3-(trifluoromethyl)phenyl]-4,5-dihydro-1,2,4-oxadiazol-3-yl}-1,2,5-oxadiazol-3-yl)amino]ethylmethanesulfonate (1.2 g, 2.9 mmol) was dissolved inN,N-dimethylformamide (2.7 mL). After sodium azide (280 mg, 4.3 mmol)was added in one portion, the temperature was brought to 65° C. and thereaction stirred for 6 hours. After cooling back to room temperature,water (10 mL) was added to quench the reaction. The product wasextracted with ethyl acetate (3×10 mL) and the combined organic layerswere dried over sodium sulfate. The solvent was removed in vacuo to givethe desired product (1.05 g, 96%). LCMS calculated for C₁₃H₁₀F₃N₆O₃(M-N₂+H)⁺: m/z=355.0. ¹H NMR (400 MHz, DMSO-d₆): δ 8.09 (s, 1H), 7.93(m, 2H), 7.79 (t, J=8.2 Hz, 1H), 6.75 (t, J=5.8 Hz, 1H), 3.52 (t, J=5.7Hz, 2H), 3.44 (q, J=5.5 Hz, 2H).

Step 6:3-{4-[(2-Aminoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-[3-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5(4H)-onehydroiodide

To a solution of3-{4-[(2-azidoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-[3-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5(4H)-one(1.05 g, 2.8 mmol) in methanol (12 mL) was added sodium iodide (2.5 g,17 mmol). After stirring for 10 minutes, a solution ofchlorotrimethylsilane (2.1 mL, 17 mmol) in methanol (1.41 mL) was addeddropwise over 15 minutes. The reaction continued to stir for 40 minutesand then a solution of sodium thiosulfate (2.7 g, 17 mmol) in water(12.5 mL) was added in one portion. A beige solid precipitated uponaddition of the sodium thiosulfate solution and it was collected byvacuum filtration. The solid was rinsed with water (2×10 mL) and wasdried under vacuum overnight to give the desired product. A solid hadalso precipitated from the filtrate and it was collected by vacuumfiltration. After washing with water (3×10 mL) in the funnel, theproduct was dried overnight under vacuum. The solid was slurry washedwith ethyl acetate (3.8 mL) for 1 hour and recollected by filtration.After rinsing with ethyl acetate (2×2 mL) and drying overnight,additional product was obtained. In total, 760 mg of desired product(57%) was obtained as the hydroiodide salt. LCMS calculated forC₁₃H₁₂F₃N₆O₃ (M+H)⁺: m/z=357.1. ¹H NMR (400 MHz, DMSO-d₆): δ 8.10 (s,1H), 7.95 (m, 2H), 7.81 (t, J=8.1 Hz, 1H), 7.68 (br s, 2H), 6.74 (t,J=6.7 Hz, 1H), 3.49 (m, 2H), 3.03 (t, J=6.7 Hz, 2H).

Step 7:4-({2-[(Aminosulfonyl)amino]ethyl}amino)-N′-hydroxy-N-[3-(trifluoromethyl)phenyl]-1,2,5-oxadiazole-3-carboximidamide

To a solution of chlorosulfonyl isocyanate (9.2 μL, 0.11 mmol) indichloromethane (0.24 mL), at 0° C. and under a nitrogen atmosphere, wasadded tert-butyl alcohol (10 μL, 0.11 mmol) in a dropwise fashion. Thesolution was allowed to stir at room temperature for 1 hour to obtain asolution of tert-butyl [chlorosulfonyl]carbamate.

In a separate flask,3-{4-[(2-aminoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-[3-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5(4H)-onehydroiodide (26 mg, 0.053 mmol) was suspended in dichloromethane (0.5mL). A nitrogen atmosphere was established and the temperature broughtto 0° C. The tert-butyl [chlorosulfonyl]carbamate solution (prepared asabove) was added over 5 minutes to the stirred suspension of the aminesalt. After 10 minutes, triethylamine (37 μL, 0.27 mmol) was addeddropwise. The reaction mixture was stirred at room temperature for 1.5hours. After concentrating in vacuo, the residue was treated withtrifluoroacetic acid (0.5 mL, 6 mmol). This was stirred for 1 hour andthe mixture was again concentrated to dryness in vacuo. The dried solidswere suspended in methanol (0.5 mL) and a 2.0 N NaOH in water (0.53 mL,1.1 mmol) was added in one portion. The reaction was heated to 45° C.and stirred for 30 minutes. After neutralization with acetic acid (60μL, 1.1 mmol), the product was purified by preparative LCMS to give thedesired product (8.5 mg, 39%). LCMS calculated for C₁₂H₁₅F₃N₇O₄S (M+H)⁺:m/z=410.0. ¹H NMR (400 MHz, CD3OD): δ 7.36 (t, J=7.8 Hz, 1H), 7.23 (d,J=7.8 Hz, 1H), 7.10 (s, 1H), 7.03 (d, J=7.8 Hz, 1H), 3.48 (m, 2H), 3.29(m, 2H).

Example 14N′-Hydroxy-4-({2-[(methylsulfonyl)amino]ethyl}amino)-N-[3-(trifluoromethyl)phenyl]-1,2,5-oxadiazole-3-carboximidamide

The title compound was prepared according to the procedure of Example32, step 5, usingN-hydroxy-4-({2-[(methylsulfonyl)amino]ethyl}amino)-1,2,5-oxadiazole-3-carboximidamideand 3-trifluoromethylaniline [Aldrich, product #A41801] as the startingmaterials. LCMS for C₁₃H₁₆F₃N₆O₄S (M+H)⁺: m/z=409.1. ¹H NMR (500 MHz,DMSO-d₆): δ 11.63 (s, 1H), 9.08 (s, 1H), 7.39 (t, J=7.6 Hz, 1H), 7.21(m, 2H), 7.10 (s, 1H), 6.99 (d, J=8.1 Hz, 1H), 6.28 (t, J=5.4 Hz, 1H),3.36 (q, J=5.8 Hz, 2H), 3.17 (q, J=5.8 Hz, 2H), 2.91 (s, 3H).

Example 154-({3-[(Aminosulfonyl)amino]propyl}amino)-N′-hydroxy-N-[3-(trifluoromethyl)phenyl]-1,2,5-oxadiazole-3-carboximidamide

Step 1:3-(4-Amino-1,2,5-oxadiazol-3-yl)-4-[3-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5(4H)-one

The desired compound was prepared according to the procedure of Example9, step 1, using4-amino-N′-hydroxy-N-[3-(trifluoromethyl)phenyl]-1,2,5-oxadiazole-3-carboximidamide[see US Pat. App. Pub. No. 2006/0258719] as the starting material in 97%yield. LCMS for C₁₁H₇F₃N₅O₃ (M+H)⁺: m/z=314.1.

Step 2:2,2,2-Trifluoro-N-(4-{5-oxo-4-[3-(trifluoromethyl)phenyl]-4,5-dihydro-1,2,4-oxadiazol-3-yl}-1,2,5-oxadiazol-3-yl)acetamide

The desired compound was prepared according to the procedure of Example9, step 2, using3-(4-amino-1,2,5-oxadiazol-3-yl)-4-[3-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5(4H)-oneas the starting material in 90% yield. LCMS for C₁₃H₆F₆N₅O₄ (M+H)⁺:m/z=410.0. ¹H NMR (400 MHz, DMSO-d₆): δ 7.91-7.88 (m, 2H), 7.76-7.69 (m,2H).

Step 3:3-{4-[(3-Methoxypropyl)amino]-1,2,5-oxadiazol-3-yl}-4-[3-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5(4H)-one

The desired compound was prepared according to the procedure of Example7, step 3, using2,2,2-trifluoro-N-(4-{5-oxo-4-[3-(trifluoromethyl)phenyl]-4,5-dihydro-1,2,4-oxadiazol-3-yl}-1,2,5-oxadiazol-3-yl)acetamideas the starting material in 49% yield. LCMS for C₁₅H₁₅F₃N₅O₄ (M+H)⁺:m/z=386.1. ¹H NMR (300 MHz, CDCl₃): δ 7.83 (d, J=8.1 Hz, 1H), 7.72-7.67(m, 2H), 7.59 (d, J=7.5 Hz, 1H), 6.08-6.04 (m, 1H), 3.57 (t, J=5.6 Hz,2H), 3.54-3.47 (m, 2H), 3.40 (s, 3H), 2.01-1.93 (m, 2H).

Step 4:3-{4-[(3-Hydroxypropyl)amino]-1,2,5-oxadiazol-3-yl}-4-[3-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5(4H)-one

The desired compound was prepared according to the procedure of Example7, step 4, using3-{4-[(3-methoxypropyl)amino]-1,2,5-oxadiazol-3-yl}-4-[3-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5(4H)-oneas the starting material in 69% yield. LCMS for C₁₄H₁₃F₃N₅O₄. (M+H)⁺:m/z=372.1. ¹H NMR (400 MHz, DMSO-d₆): δ 8.07 (s, 1H), 7.95-7.90 (m, 2H),7.79 (dd, J=7.9, 7.9 Hz, 1H), 6.55 (t, J=5.6 Hz, 1H), 4.59 (t, J=5.1 Hz,1H), 3.47-3.42 (m, 2H), 3.30-3.25 (m, 2H), 1.72-1.65 (m, 2H).

Step 5:3-{4-[(3-Azidopropyl)amino]-1,2,5-oxadiazol-3-yl}-4-[3-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5(4H)-one

The desired compound was prepared according to the procedure of Example7, step 5, using3-{4-[(3-hydroxypropyl)amino]-1,2,5-oxadiazol-3-yl}-4-[3-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5(4H)-oneas the starting material in 92% yield. LCMS for C₁₄H₁₁F₃N₈O₃Na (M+Na)±:m/z=419.0.

Step 6:3-{4-[(3-Aminopropyl)amino]-1,2,5-oxadiazol-3-yl}-4-[3-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5(4H)-onehydroiodide

The desired compound was prepared according to the procedure of Example7, step 6, using3-{4-[(3-azidopropyl)amino]-1,2,5-oxadiazol-3-yl}-4-[3-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5(4H)-oneas the starting material in 92% yield. LCMS for C₁₄H₁₄F₃N₆O₃ (M+H)⁺:m/z=371.1. ¹H NMR (400 MHz, DMSO-d₆): δ 8.09 (s, 1H), 7.96-7.92 (m, 2H),7.80 (dd, J=8.0, 7.8 Hz, 1H), 7.53 (br s, 2H), 6.70-6.65 (m, 1H), 4.10(br s, 1H), 3.32-3.31 (m, 2H), 2.81-2.78 (m, 2H), 1.85-1.82 (m, 2H).

Step 7:4-({3-[(Aminosulfonyl)amino]propyl}amino)-N′-hydroxy-N-[3-(trifluoromethyl)phenyl]-1,2,5-oxadiazole-3-carboximidamide

A solution of3-{4-[(3-aminopropyl)amino]-1,2,5-oxadiazol-3-yl}-4-[3-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5(4H)-onehydroiodide (1.5 g, 3.0 mmol) and sulfamide (1.7 g, 18 mmol) in pyridine(60 mL) was heated in a microwave at 130° C. for 10 min. The reactionmixture was concentrated to give the crude intermediateN-{3-[(4-{5-oxo-4-[3-(trifluoromethyl)phenyl]-4,5-dihydro-1,2,4-oxadiazol-3-yl}-1,2,5-oxadiazol-3-yl)amino]propyl}sulfamide.A solution of the crude intermediate in methanol (90 mL) was treatedwith 2 N NaOH (12 mL, 24 mmol) and stirred at 25° C. for 30 min. Thereaction mixture was treated with 6 M HCl until the solution was acidicand extracted with ethyl acetate (250 mL). The organic layer was washedwith water (100 mL) and brine (100 mL), dried over anhydrous sodiumsulfate, filtered, and concentrated to give a crude residue. Thismaterial was purified by preparative LCMS to give the desired product(1.1 g, 82%) as a gummy solid. LCMS for C₁₃H₁₇F₃N₇O₄S (M+H)⁺: m/z=424.0.¹H NMR (400 MHz, DMSO-d₆): δ 11.6 (s, 1H), 9.12 (s, 1H), 7.37 (dd,J=8.0, 8.0 Hz, 1H), 7.21-7.18 (m, 1H), 7.07 (s, 1H), 6.95 (d, J=10.0 Hz,1H), 6.52 (br s, 3H), 6.17 (t, J=6.0 Hz, 1H), 3.28-3.22 (m, 2H),2.93-2.89 (m, 2H), 1.77-1.73 (m, 2H).

Example 16N′-Hydroxy-4-({3-[(methylsulfonyl)amino]propyl}amino)-N-[3-(trifluoromethyl)phenyl]-1,2,5-oxadiazole-3-carboximidamide

A solution of3-{4-[(3-aminopropyl)amino]-1,2,5-oxadiazol-3-yl}-4-[3-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5(4H)-onehydroiodide (from Example 19, step 6; 25 mg, 50 μmol) in dichloromethane(1 mL) was treated with triethylamine (17 μL, 0.12 mmol) andmethanesulfonyl chloride (6 μL, 70 μmol) and stirred at 25° C. for 2 h.The reaction mixture was concentrated to give the intermediate,N-{3-[(4-{5-oxo-4-[3-(trifluoromethyl)phenyl]-4,5-dihydro-1,2,4-oxadiazol-3-yl}-1,2,5-oxadiazol-3-yl)amino]propyl}methanesulfonamide,as a crude residue which was used without further purification. Asolution of the crude intermediate in methanol (1 mL) was treated with 2N NaOH (0.25 mL, 0.5 mmol) and stirred at 25° C. for 30 min. Thereaction mixture was treated with acetic acid (50 μL, 0.9 mmol),filtered and purified by preparative LCMS to give the desired product(13 mg, 65%) as a solid. LCMS for C₁₄H₁₈F₃N₆O₄S (M+H)⁺: m/z=423.1. ¹HNMR (400 MHz, DMSO-d₆): δ 11.6 (s, 1H), 9.11 (s, 1H), 7.37 (dd, J=8.0,8.0 Hz, 1H), 7.20 (d, J=7.8 Hz, 1H), 7.07-7.01 (m, 2H), 6.96 (d, J=8.0Hz, 1H), 6.20 (t, J=5.9 Hz, 1H), 3.27-3.22 (m, 2H), 2.99-2.94 (m, 2H),2.87 (s, 3H), 1.78-1.71 (m, 2H).

Example 17N-(4-Fluoro-3-methylphenyl)-N′-hydroxy-4-({2-[(methylsulfonyl)amino]ethyl}amino)-1,2,5-oxadiazole-3-carboximidamide

Step 1: tert-Butyl {2-[(methylsulfonyl)amino]ethyl}carbamate

N-(2-Aminoethyl)(tert-butoxy)carboxamide (17.5 mL, 0.11 mol) [Alfa#L19947] was stirred in dichloromethane (320 mL) and triethylamine (33mL, 0.24 mol) was added. A solution of methanesulfonyl chloride (8.5 mL,0.11 mol) in dichloromethane (10 mL) was added. The resulting mixturewas stirred for 1 hour and water (30 mL) was added. The product wasextracted with dichloromethane (3×30 mL), dried over sodium sulfate andconcentrated in vacuo to give the desired product (21 g, 81%). LCMScalculated for C₃H₁₁N₂O₂S (M-Boc+H)⁺: m/z=139.1.

Step 2: N-(2-Aminoethyl)methanesulfonamide dihydrochloride

tert-Butyl {2-[(methylsulfonyl)amino]ethyl}carbamate (21 g, 88 mmol) wasstirred in a solution of 4 N hydrogen chloride in 1,4-dioxane (97 mL,388 mmol) for 30 minutes. Trituration with ethyl acetate and hexanesfollowed by diethyl ether and hexanes gave the desired compound as a gum(19 g, 100%). LCMS calculated for C₃H₁₁N2O2S (M+H)⁺: m/z=139.0.

Step 3:4-Amino-N′-hydroxy-N-{2-[(methylsulfonyl)amino]ethyl}-1,2,5-oxadiazole-3-carboximidamide

4-Amino-N-hydroxy-1,2,5-oxadiazole-3-carboximidoyl chloride (9.7 g, 60mmol) was stirred in ethanol (460 mL) andN-(2-aminoethyl)methanesulfonamide dihydrochloride (19 g, 109 mmol) wasadded slowly in portions and the temperature rose to 25° C. Aftercooling back to 0° C., triethylamine (53 mL, 380 mmol) was addeddropwise over 15 minutes and the reaction was stirred for an additional15 minutes. The solution was washed with water (300 mL) and brine (300mL). The organic layer was dried over sodium sulfate and concentrated invacuo to give the desired product (16 g, 100%). LCMS calculated forC₆H₁₃N₆O₄S (M+H)⁺: m/z=265.1. ¹H NMR (400 MHz, DMSO-d₆): δ 10.16 (s,1H), 9.07 (m, 1H), 7.18 (m, 1H), 6.37 (s, 2H), 3.36 (m, 2H), 3.15 (m,2H), 2.87 (s, 3H).

Step 4:N′-Hydroxy-4-({2-[(methylsulfonyl)amino]ethyl}amino)-1,2,5-oxadiazole-3-carboximidamide

4-Amino-N′-hydroxy-N-{2-[(methylsulfonyl)amino]ethyl}-1,2,5-oxadiazole-3-carboximidamide(0.47 g, 1.8 mmol) was stirred in 1,2-ethanediol (38 mL). Potassiumhydroxide (600 g, 11 mmol) was added in one portion. The reaction washeated at 130° C. for 4 hours and allowed to cool to room temperature. 1N HCl solution (60 mL) was added and the product was extracted withethyl acetate (4×40 mL). The combined organics were dried over sodiumsulfate and concentrated in vacuo to give the desired product (0.45 g,96%). LCMS calculated for C₆H₁₂N₆O₄S (M+H)⁺: m/z=265.1. ¹H NMR (400 MHz,DMSO-d₆): δ 10.49 (s, 1H), 7.18 (m, 1H), 6.20 (m, 3H), 3.36 (m, 2H),3.15 (m, 2H), 2.87 (s, 3H).

Step 5:N-(4-Fluoro-3-methylphenyl)-N′-hydroxy-4-({2-[(methylsulfonyl)amino]ethyl}amino)-1,2,5-oxadiazole-3-carboximidamide

N-Hydroxy-4-({2-[(methyl sulfonyl)amino]ethyl}amino)-1,2,5-oxadiazole-3-carboximidamide (35 mg, 0.13 mmol) wasstirred in 1,4-dioxane (2 mL) and 6 N hydrogen chloride solution (4 mL)was added. The solution was cooled to 0° C. and a solution of sodiumnitrite (11 mg, 0.16 mmol) in water (3 mL) was slowly added. The mixturewas stirred for 1 hour at 0° C. and evaporated. Dry 1,4-dioxane (2 mL)was added and the mixture evaporated two more times. A solution of4-fluoro-3-methylaniline [Aldrich, product #559415] (25 mg, 0.20 mmol)in ethanol (2 mL) was added and the mixture was stirred for 1 hour.Purification by preparative LCMS (pH 2) gave the desired compound (17mg, 27%). LCMS calculated for C₁₃H₁₈FN₆O₄S (M+H)⁺: m/z=373.1. ¹H NMR(400 MHz, DMSO-d₆): δ 11.25 (s, 1H), 8.61 (s, 1H), 7.18 (m, 1H), 6.91(m, 1H), 6.72 (m, 1H), 6.58 (m, 1H), 6.24 (s, 1H), 3.32 (m, 2H), 3.11(m, 2H), 2.89 (s, 3H), 2.05 (s, 3H).

Example 184-({2-[(Aminosulfonyl)amino]ethyl}amino)-N-(3-cyano-4-fluorophenyl)-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide

Step 1:N-(3-Cyano-4-fluorophenyl)-N′-hydroxy-4-[(2-methoxyethyl)amino]-1,2,5-oxadiazole-3-carboximidamide

The desired compound was prepared according to the procedure of Example17, step 1, usingN-hydroxy-4-[(2-methoxyethyl)amino]-1,2,5-oxadiazole-3-carboximidoylchloride [made according to Example 5, steps 1 through 5] and5-amino-2-fluorobenzonitrile [Aldrich, product #639877] as the startingmaterials in 100% yield. LCMS for C₁₃H₁₄FN₆O₃ (M+H)⁺: m/z=321.0.

Step 2:2-Fluoro-5-[3-{4-[(2-methoxyethyl)amino]-1,2,5-oxadiazol-3-yl}-5-oxo-1,2,4-oxadiazol-4(5H)-yl]benzonitrile

The desired compound was prepared according to the procedure of Example17, step 2, usingN-(3-cyano-4-fluorophenyl)-N′-hydroxy-4-[(2-methoxyethyl)amino]-1,2,5-oxadiazole-3-carboximidamideas the starting material in 91% yield. LCMS for C₁₄H₁₂FN₆O₄ (M+H)⁺:m/z=347.0. ¹H NMR (400 MHz, DMSO-d₆): δ 8.25 (dd, J=5.7, 2.6 Hz, 1H),8.06 (m, 1H), 7.77 (t, J=9.2 Hz, 1H), 6.41 (t, J=5.7 Hz, 1H), 3.48 (m,2H), 3.40 (q, J=5.4 Hz, 2H), 3.25 (s, 3H).

Step 3:2-Fluoro-5-[3-{4-[(2-hydroxyethyl)amino]-1,2,5-oxadiazol-3-yl}-5-oxo-1,2,4-oxadiazol-4(5H)-yl]benzonitrile

The desired compound was prepared according to the procedure of Example17, step 3, using2-fluoro-5-[3-{4-[(2-methoxyethyl)amino]-1,2,5-oxadiazol-3-yl}-5-oxo-1,2,4-oxadiazol-4(5H)-yl]benzonitrileas the starting material in quantitative yield. LCMS for C₁₃H₁₀FN₆O₄(M+H)⁺: m/z=333.0.

Step 4:2-({4-[4-(3-Cyano-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}amino)ethylmethanesulfonate

The desired compound was prepared according to the procedure of Example17, step 4, using2-fluoro-5-[3-{4-[(2-hydroxyethyl)amino]-1,2,5-oxadiazol-3-yl}-5-oxo-1,2,4-oxadiazol-4(5H)-yl]benzonitrileas the starting material in 88% yield. LCMS for C₁₄H₁₂FN₆O₆S (M+H)⁺:m/z=411.0.

Step 5:5-[3-{4-[(2-Azidoethyl)amino]-1,2,5-oxadiazol-3-yl}-5-oxo-1,2,4-oxadiazol-4(5H)-yl]-2-fluorobenzonitrile

The desired compound was prepared according to the procedure of Example17, step 5, using2-({4-[4-(3-cyano-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}amino)ethylmethanesulfonate as the starting material in 95% yield.

Step 6:5-[3-{4-[(2-Aminoethyl)amino]-1,2,5-oxadiazol-3-yl}-5-oxo-1,2,4-oxadiazol-4(5H)-yl]-2-fluorobenzonitrilehydroiodide

The desired compound was prepared according to the procedure of Example17, step 6, using5-[3-{4-[(2-azidoethyl)amino]-1,2,5-oxadiazol-3-yl}-5-oxo-1,2,4-oxadiazol-4(5H)-yl]-2-fluorobenzonitrileas the starting material in 57% yield. LCMS for C₁₃H₁₁FN₇O₃ (M+H)⁺:m/z=332.0. ¹H NMR (400 MHz, DMSO-d₆): δ 8.29 (dd, J=5.8, 2.7 Hz, 1H),8.09 (m, 1H), 7.83 (br s, 3H), 7.79 (t, J=9.0 Hz, 1H), 6.77 (t, J=5.9Hz, 1H), 3.50 (q, J=6.4 Hz, 2H), 3.04 (m, 2H).

Step 7:4-({2-[(Aminosulfonyl)amino]ethyl}amino)-N-(3-cyano-4-fluorophenyl)-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide

In a microwave vial,5-[3-{4-[(2-aminoethyl)amino]-1,2,5-oxadiazol-3-yl}-5-oxo-1,2,4-oxadiazol-4(5H)-yl]-2-fluorobenzonitrilehydroiodide (20.0 mg, 0.044 mmol) and sulfamide (25 mg, 0.26 mmol) weresuspended in pyridine (0.5 mL). The reaction was heated to 120° C. for10 minutes in a microwave reactor. The solvent was removed and theresidue dissolved in methanol (0.17 mL). A solution of 2.0 N NaOH inwater (0.22 mL, 0.44 mmol) was added in one portion. The reaction wasstirred at room temperature overnight. After neutralization with aceticacid (50 μL), the product was purified using preparative LCMS to givethe title compound (4.9 mg, 29%). LCMS for C₁₂H₁₄FN₈O₄S (M+H)⁺:m/z=385.0. ¹H NMR (400 MHz, DMSO-d₆): δ 11.65 (s, 1H), 9.08 (s, 1H),7.34 (t, J=9.1 Hz, 1H), 7.22 (dd, J=5.4, 2.8 Hz, 1H), 7.13 (m, 1H), 6.70(t, J=5.9 Hz, 1H), 6.59 (s, 2H), 6.20 (t, J=6.1 Hz, 1H), 3.34 (m, 2H),3.09 (m, 2H).

Example 19N-(3-Cyano-4-fluorophenyl)-N′-hydroxy-4-({2-[(methylsulfonyl)amino]ethyl}amino)-1,2,5-oxadiazole-3-carboximidamide

The title compound was prepared according to the procedure of Example21, step 5, usingN-hydroxy-4-({2-[(methylsulfonyl)amino]ethyl}amino)-1,2,5-oxadiazole-3-carboximidamideand 3-cyano-4-fluoroaniline [Aldrich, product #639877] as the startingmaterials. LCMS for C₁₃H₁₄FN₇NaO₄S (M+Na)⁺: m/z=406.0. ¹H NMR (400 MHz,DMSO-d₆): δ 11.65 (s, 1H), 9.08 (s, 1H), 7.35 (m, 1H), 7.18 (m, 3H),6.56 (m, 1H), 6.23 (m, 1H), 6.24 (s, 2H), 3.32 (m, 2H), 3.14 (m, 2H),2.89 (s, 3H).

Example 204-({2-[(Aminosulfonyl)amino]ethyl}amino)-N-[(4-bromo-2-furyl)methyl]-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide

Step 1: tert-Butyl [(4-bromo-2-furyl)methyl]carbamate

4-Bromo-2-furaldehyde [Aldrich, product #666599] (10.0 g, 57.1 mmol) wasdissolved in ethanol (50 mL) and water (50 mL). N-Hydroxyaminehydrochloride (7.15 g, 103 mmol) and sodium acetate (8.44 g, 103 mmol)were added sequentially and the reaction mixture was brought to refluxat 100° C. for 1 hour. The solution was partially concentrated and theprecipitate was collected and washed with cold water (2×10 mL). Thefiltrate was extracted with ethyl acetate (3×25 mL) and the combinedorganic layers were washed with brine (50 mL). After drying over sodiumsulfate, the solution was concentrated in vacuo. The residue wascombined with the precipitate and dissolved in acetic acid (70 mL).After placing in an ice-bath, zinc (14.7 g, 225 mmol) was addedportion-wise over 25 minutes. The reaction warmed to room temperatureover 1.5 hours and was filtered through Celite. The solvent was removedin vacuo.

The residue was stirred in tetrahydrofuran (72 mL). A solution of 2.0 NNaOH in water (179 mL, 358 mmol) was added dropwise over 45 minutes.After 5 minutes, di-tert-butyldicarbonate (16.9 g, 77.4 mmol) was addeddropwise. The reaction was stirred for 2 hours and the tetrahydrofuranwas removed in vacuo. Ethyl acetate (100 mL) was added and thesuspension was filtered. The organic layer was collected and the productextracted with ethyl acetate (2×50 mL). The combined organic layers werewashed with brine (100 mL) and water (100 mL), dried over sodium sulfateand concentrated in vacuo to give the desired product (15.3 g, 79%).LCMS calculated for C₁₀H₁₄BrNNaO₃ (M+Na)⁺: m/z=298.0. ¹H NMR (400 MHz,DMSO-d₆): δ 7.79 (s, 1H), 7.37 (t, J=5.8 Hz, 1H), 6.33 (s, 1H), 4.06 (d,J=6.1 Hz, 2H), 1.36 (s, 9H).

Step 2: 1-(4-Bromo-2-furyl)methanamine trifluoroacetate

Under a nitrogen atmosphere, a solution of tert-butyl[(4-bromo-2-furyl)methyl]carbamate (15.3 g, 55.4 mmol) indichloromethane (86 mL) at 0° C. was treated with trifluoroacetic acid(43 mL) over 15 minutes. The reaction mixture warmed to room temperatureover 30 minutes. The solvent was removed in vacuo and chased withtoluene (3×50 mL). The product was lyophilized for 18 hours to give thedesired product as a brown solid (13.0 g, 81%). LCMS calculated forC₅H₄BrO (M-NH₂)⁺: m/z=158.9, 160.9. ¹H NMR (400 MHz, DMSO-d₆): δ 8.34(br s, 3H), 8.01 (s, 1H), 6.70 (s, 1H), 4.08 (s, 1H).

Step 3:N-[(4-Bromo-2-furyl)methyl]-N′-hydroxy-4-[(2-methoxyethyl)amino]-1,2,5-oxadiazole-3-carboximidamide

N-Hydroxy-4-(2-methoxyethylamino)-1,2,5-oxadiazole-3-carbimidoylchloride [prepared according to the procedure of Example 5, steps 1through 5] (4.5 g, 20.3 mmol) was stirred in ethanol (20 mL) at roomtemperature. To this, a solution of 1-(4-bromo-2-furyl)methanaminetrifluoroacetate (6.5 g, 22.4 mmol) in ethanol (24 mL) was added and themixture was stirred for 15 minutes. Triethylamine (6.3 mL, 44.8 mmol)was added dropwise over 10 minutes and the reaction was stirred for anadditional 15 minutes. The solvent was removed in vacuo and after addingwater (50 mL), the product was extracted with ethyl acetate (3×50 mL).The combined organic layers were washed with brine (50 mL), dried oversodium sulfate and concentrated to give the desired product (7.5 g,100%). LCMS calculated for C₁₁H₁₅BrN₅O₄ (M+H)⁺: m/z=359.9, 361.9.

Step 4:4-[(4-Bromo-2-furyl)methyl]-3-{4-[(2-methoxyethyl)amino]-1,2,5-oxadiazol-3-yl}-1,2,4-oxadiazol-5(4H)-one

N-[(4-Bromo-2-furyl)methyl]-N′-hydroxy-4-[(2-methoxyethyl)amino]-1,2,5-oxadiazole-3-carboximidamide(7.3 g, 20.4 mmol) and 1,1′-carbonyldiimidazole (5.0 g, 30.5 mmol) weredissolved in ethyl acetate (72 mL). The reaction mixture was heated at65° C. for 15 minutes. Ethyl acetate (70 mL) was added and the crudereaction was washed with 0.1 N hydrogen chloride in water (2×70 mL). Theorganic layer was dried over sodium sulfate and concentrated in vacuo.Purification by flash chromatography on silica gel with an eluent ofethyl acetate in hexanes gave the desired product (4.1 g, 90%). LCMScalculated for C₁₂H₁₃BrN₅O₅ (M+H)⁺: m/z=386.0, 388.0. ¹H NMR (400 MHz,CD3OD): δ 7.88 (s, 1H), 6.67 (s, 1H), 6.39 (t, J=5.7 Hz, 1H), 5.07 (s,2H), 3.50 (m, 2H), 3.41 (q, J=5.7 Hz, 2H), 3.25 (s, 3H).

Step 5:4-[(4-Bromo-2-furyl)methyl]-3-{4-[(2-hydroxyethyl)amino]-1,2,5-oxadiazol-3-yl}-1,2,4-oxadiazol-5(4H)-one

In a round bottom flask under nitrogen atmosphere,4-[(4-bromo-2-furyl)methyl]-3-{4-[(2-methoxyethyl)amino]-1,2,5-oxadiazol-3-yl}-1,2,4-oxadiazol-5(4H)-one(8.2 g, 21 mmol) was stirred in dichloromethane (68 mL). The temperaturewas brought to −78° C. and a solution of 1.0 M boron tribromide indichloromethane (43 mL, 43 mmol) was added dropwise over 45 minutes. Thereaction stirred at −78° C. for 45 minutes and continued to stir at 0°C. for an additional 30 minutes. While remaining at 0° C., a saturatedsolution of sodium bicarbonate in water (120 mL) was added dropwise over25 minutes.

After warming to room temperature, the organic layer was collected andthe aqueous layer was extracted with ethyl acetate (2×50 mL). Thecombined organic layers were washed with brine (100 mL), dried oversodium sulfate and concentrated in vacuo to give the desired product(7.7 g, 97%) along with a small amount of3-{4-[(2-bromoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-[(4-bromo-2-furyl)methyl]-1,2,4-oxadiazol-5(4H)-one.LCMS calculated for C₁₁H₁₁BrN₅O₅ (M+H)⁺: m/z=371.7, 374.0. ¹H NMR (400MHz, DMSO-d₆): δ 7.89 (s, 1H), 6.68 (s, 1H), 6.31 (t, J=5.8 Hz, 1H),5.08 (s, 2H), 4.85 (br s, 1H), 3.56 (m, 2H), 3.30 (q, J=5.6 Hz, 2H).

Step 6:2-[(4-{4-[(4-Bromo-2-furyl)methyl]-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl}-1,2,5-oxadiazol-3-yl)amino]ethylmethanesulfonate

To a solution of4-[(4-bromo-2-furyl)methyl]-3-{4-[(2-hydroxyethyl)amino]-1,2,5-oxadiazol-3-yl}-1,2,4-oxadiazol-5(4H)-one(7.7 g, 21 mmol, containing also some of the correspondingbromo-compound) in ethyl acetate (100 mL) was added methanesulfonylchloride (0.96 mL, 12 mmol) in one portion. The reaction was stirred for5 minutes and triethylamine (1.6 mL, 11 mmol) was added, also in oneportion. After stirring for 30 minutes, additional methanesulfonylchloride (0.4 mL, 5 mmol) was added, followed 5 minutes later bytriethylamine (0.58 mL, 4.2 mmol). After 15 minutes, the reaction wasquenched with the addition of water (100 mL). The product was extractedwith ethyl acetate (3×50 mL) and the combined organic layers washed withbrine (100 mL). After drying over sodium sulfate, the solvent wasremoved in vacuo to give the desired product (9.3 g, 100%). LCMScalculated for C₁₂H₁₃BrN₅O₇S (M+H)⁺: m/z=449.8, 451.8. ¹H NMR (300 MHz,DMSO-d₆): δ 7.88 (s, 1H), 6.73 (t, J=6.2 Hz, 1H), 6.68 (s, 1H), 5.08 (s,2H), 4.37 (m, 2H), 3.59 (q, J=5.8 Hz, 2H), 3.16 (s, 3H).

Step 7:3-{4-[(2-Azidoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-[(4-bromo-2-furyl)methyl]-1,2,4-oxadiazol-5(4H)-one

2-[(4-{4-[(4-Bromo-2-furyl)methyl]-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl}-1,2,5-oxadiazol-3-yl)amino]ethylmethanesulfonate (9.1 g, 20 mmol, containing also some of thecorresponding bromo-compound) was dissolved in dimethylformamide (90mL). Sodium azide (1.97 g, 30.3 mmol) was added in one portion and after5 minutes, the temperature was brought to 65° C. The reaction stirredfor 2 hours and was allowed to cool back to room temperature. Water (200mL) was added to quench the reaction. The product was extracted withethyl acetate (3×100 mL) and the combined organic layers were washedwith brine (2×150 mL) and water (150 mL). After drying over sodiumsulfate, the solvent was removed in vacuo to give the desired product(7.7 g, 96%). LCMS calculated for C₁₁H₉BrN₈NaO₄ (M+Na)⁺: m/z=418.7,421.0.¹H NMR (400 MHz, DMSO-d₆): δ 7.88 (s, 1H), 6.71 (t, J=5.7 Hz, 1H),6.68 (s, 1H), 5.08 (s, 2H), 3.54 (t, J=5.7 Hz, 2H), 3.47 (q, J=5.7 Hz,2H).

Step 8:3-{4-[(2-Aminoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-[(4-bromo-2-furyl)methyl]-1,2,4-oxadiazol-5(4H)-onehydroiodide

To a solution of3-{4-[(2-azidoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-[(4-bromo-2-furyl)methyl]-1,2,4-oxadiazol-5(4H)-one(7.7 g, 19 mmol) in methanol (80 mL) was added sodium iodide (17.4 g,116 mmol). After stirring for 10 minutes, a solution ofchlorotrimethylsilane (14.8 mL, 116 mmol) was added dropwise over 5minutes. The reaction continued to stir for 1 hour, at which time it wasslowly added to a solution of sodium thiosulfate (23.0 g, 145 mmol) inwater (800 mL) at 0° C., resulting in a precipitate. The flask wasrinsed with methanol (10 mL) and the precipitate was collected throughvacuum filtration. The solid was rinsed with cold water (2×25 mL) andwas dried under vacuum to give the desired product (5.8 g, 60%) as thehydroiodide salt. LCMS calculated for C₁₁H₁₂BrN₆O₄ (M+H)⁺: m/z=370.9,372.8. ¹H NMR (400 MHz, DMSO-d₆): δ 7.86 (s, 1H), 7.36 (br s, 3H), 6.68(t, J=5.8 Hz, 1H), 6.65 (s, 1H), 5.07 (s, 2H), 3.45 (q, J=5.8 Hz, 2H),2.98 (t, J=5.8 Hz, 2H).

Step 9:4-({2-[(Aminosulfonyl)amino]ethyl}amino)-N-[(4-bromo-2-furyl)methyl]-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide

In a microwave vial,3-{4-[(2-aminoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-[(4-bromo-2-furyl)methyl]-1,2,4-oxadiazol-5(4H)-onehydroiodide (30 mg, 0.060 mmol) and sulfamide (29 mg, 0.30 mmol) weresuspended in pyridine (1 mL). The reaction mixture was flushed withnitrogen and heated at 130° C. for 3 minutes in a microwave reactor. Thesolvent was removed and the crude intermediate was suspended in methanol(1 mL). A 2.0 N solution of NaOH in water (0.30 mL, 0.60 mmol) was addedin one portion and the reaction was heated to 45° C. for 30 minutes.After neutralization with acetic acid (68 μL, 1.2 mmol), the product waspurified by preparative LCMS to give the desired product (10.4 mg, 41%).LCMS calculated for C₁₀H₁₅BrN₇O₅S (M+H)⁺: m/z=423.9, 426.0. ¹H NMR (400MHz, DMSO-d₆): δ 10.87 (s, 1H), 7.75 (s, 1H), 6.83 (t, J=7.3 Hz, 1H),6.68 (t, J=6.0 Hz, 1H), 6.56 (s, 2H), 6.30 (t, J=6.0 Hz, 1H), 6.23 (s,1H), 4.56 (d, J=7.0 Hz, 2H), 3.32 (q, J=6.3 Hz, 2H), 3.07 (q, J=6.3 Hz,2H).

Example 214-({2-[(Aminosulfonyl)amino]ethyl}amino)-N-[(4-chloro-2-furyl)methyl]-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide

Step 1: 4-Chloro-2-furaldehyde

To a stirred suspension of aluminum trichloride (29.8 g, 0.223 mol) indichloromethane (200 mL) under nitrogen atmosphere was added2-furancarboxaldehyde (8.44 mL, 0.102 mol) over 15 minutes. Afterstirring for 30 minutes, chlorine was bubbled into the suspension usinga pipette over a time period of 50 minutes. The flask was sealed andleft to stir at room temperature for 90 hours. The reaction mixture wasslowly added to a mixture of ice (500 mL) in a solution of 1.0 Nhydrogen chloride in water (300 mL). The mixture was left to warm toroom temperature over the next hour. The layers were separated and theorganic layer collected. Additional product was extracted withdichloromethane (2×200 mL). The combined organic layers were washed withwater (250 mL) and dried over sodium sulfate. The solvent was removed invacuo to give a crude mixture containing the desired product (14.0 g,100%, 60% purity). ¹H NMR (400 MHz, DMSO-d₆): δ 9.56 (s, 1H), 8.36 (s,1H), 7.71 (s, 1H).

Step 2: tert-Butyl [(4-chloro-2-furyl)methyl]carbamate

4-Chloro-2-furaldehyde (14.0 g, 60% purity, 64 mmol) was dissolved inethanol (50 mL) and water (50 mL). N-Hydroxyamine hydrochloride (12.6 g,182 mmol) and sodium acetate (14.9 g, 182 mmol) were added sequentiallyand the reaction mixture was brought to reflux at 100° C. for 1 hour.The solution was partially concentrated then water (25 mL) and ethylacetate (50 mL) were added. The organic layer was collected and theaqueous was extracted with ethyl acetate (2×25 mL). The combined organiclayers were washed with brine (50 mL) and water (50 mL). After dryingover sodium sulfate, the solution was concentrated in vacuo. Theintermediate was suspended in acetic acid (115 mL). The solution wascooled in an ice-bath and zinc (33.1 g, 506 mmol) was added portion-wiseover 20 minutes. The reaction warmed to room temperature over 2 hoursand was filtered through Celite. The solvent was removed in vacuo.

The residue was stirred in tetrahydrofuran (100 mL). A solution of 2.0 MNaOH in water (152 mL, 304 mmol) was added dropwise over 30 minutes. Thereaction mixture was placed in an ice-bath and after 5 minutes,di-tert-butyldicarbonate (24.3 g, 111 mmol) was added dropwise over 15minutes. The reaction was allowed to warm to room temperature over thenext 2 hours and the tetrahydrofuran was then removed in vacuo. Ethylacetate (100 mL) was added and the suspension was filtered. The organiclayer was collected and the aqueous layer extracted with ethyl acetate(2×100 mL). The combined organic layers were washed with a 1:1 mixtureof water/brine (100 mL), dried over sodium sulfate and concentrated invacuo. Purification by flash chromatography on silica gel with an eluentof ethyl acetate in hexanes gave the desired product (3.05 g, 22%). LCMScalculated for C₁₀H₁₄ClNNaO₃ (M+Na)⁺: m/z=253.9. ¹H NMR (400 MHz,DMSO-d₆): δ 7.81 (s, 1H), 7.37 (t, J=5.3 Hz, 1H), 6.32 (s, 1H), 4.05 (d,J=6.0 Hz, 2H), 1.36 (s, 9H).

Step 3: 1-(4-Chloro-2-furyl)methanamine trifluoroacetate

The desired compound was prepared according to the procedure of Example24, step 2, using tert-butyl [(4-chloro-2-furyl)methyl]carbamate as thestarting material in quantitative yield. LCMS calculated for C₅H₄ClO(M-NH₂)⁺: m/z=115.0. ¹H NMR (400 MHz, DMSO-d₆): δ 8.29 (br s, 3H), 8.04(s, 1H), 6.69 (s, 1H), 4.07 (s, 2H).

Step 4:N-[(4-Chloro-2-furyl)methyl]-N′-hydroxy-4-[(2-methoxyethyl)amino]-1,2,5-oxadiazole-3-carboximidamide

The desired compound was prepared according to the procedure of Example24, step 3, usingN-hydroxy-4-(2-methoxyethylamino)-1,2,5-oxadiazole-3-carbimidoylchloride and 1-(4-chloro-2-furyl)methanamine trifluoroacetate as thestarting material in quantitative yield. LCMS calculated forC₁₁H₁₅ClN₅O₄ (M+H)⁺: m/z=316.0.

Step 5:4-[(4-Chloro-2-furyl)methyl]-3-{4-[(2-methoxyethyl)amino]-1,2,5-oxadiazol-3-yl}-1,2,4-oxadiazol-5(4H)-one

The desired compound was prepared according to the procedure of Example24, step 4, usingN-[(4-chloro-2-furyl)methyl]-N′-hydroxy-4-[(2-methoxyethyl)amino]-1,2,5-oxadiazole-3-carboximidamideas the starting material in 51% yield. LCMS calculated for C₁₂H₁₃ClN₅O₅(M+H)⁺: m/z=342.0.

Step 6:4-[(4-Chloro-2-furyl)methyl]-3-{4-[(2-hydroxyethyl)amino]-1,2,5-oxadiazol-3-yl}-1,2,4-oxadiazol-5(4H)-one

The desired compound was prepared according to the procedure of Example24, step 5, using4-[(4-chloro-2-furyl)methyl]-3-{4-[(2-methoxyethyl)amino]-1,2,5-oxadiazol-3-yl}-1,2,4-oxadiazol-5(4H)-oneas the starting material in quantitative yield. LCMS calculated forC₁₁H₁₀ClN₅NaO₅ (M+Na)⁺: m/z=349.9.

Step 7:2-[(4-{4-[(4-Chloro-2-furyl)methyl]-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl}-1,2,5-oxadiazol-3-yl)amino]ethylmethanesulfonate

The desired compound was prepared according to the procedure of Example24, step 6, using4-[(4-chloro-2-furyl)methyl]-3-{4-[(2-hydroxyethyl)amino]-1,2,5-oxadiazol-3-yl}-1,2,4-oxadiazol-5(4H)-oneas the starting material in 69% yield. LCMS calculated for C₁₂H₁₃C₁N₅O₇S(M+H)⁺: m/z=405.8.

Step 8:3-{4-[(2-Azidoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-[(4-chloro-2-furyl)methyl]-1,2,4-oxadiazol-5(4H)-one

The desired compound was prepared according to the procedure of Example24, step 7, using2-[(4-{4-[(4-chloro-2-furyl)methyl]-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl}-1,2,5-oxadiazol-3-yl)amino]ethylmethanesulfonate as the starting material in quantitative yield. LCMScalculated for C₁₁H₉ClN₈NaO₄ (M+Na)⁺: m/z=374.9.

Step 9:3-{4-[(2-Aminoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-[(4-chloro-2-furyl)methyl]-1,2,4-oxadiazol-5(4H)-onehydroiodide

The desired compound was prepared according to the procedure of Example24, step 8, using3-{4-[(2-azidoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-[(4-chloro-2-furyl)methyl]-1,2,4-oxadiazol-5(4H)-oneas the starting material in 57% yield. LCMS calculated for C₁₁H₁₂ClN₆O₄(M+H)⁺: m/z=326.9.

Step 10:4-({2-[(Aminosulfonyl)amino]ethyl}amino)-N-[(4-chloro-2-furyl)methyl]-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide

The desired compound was prepared according to the procedure of Example24, step 9, using3-{4-[(2-aminoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-[(4-chloro-2-furyl)methyl]-1,2,4-oxadiazol-5(4H)-onehydroiodide as the starting material in 53% yield. LCMS calculated forC₁₀H₁₅ClN₇O₅S (M+H)⁺: m/z=379.9. ¹H NMR (400 MHz, DMSO-d₆): δ 10.88 (s,1H), 7.77 (s, 1H), 6.83 (t, J=6.8 Hz, 1H), 6.68 (t, J=5.9 Hz, 1H), 6.56(s, 2H), 6.30 (t, J=5.9 Hz, 1H), 6.22 (s, 1H), 4.55 (d, 2H), 3.32 (q,J=6.3 Hz, 2H), 3.06 (q, J=6.3 Hz, 2H).

Example 22 Alternate Preparation of the Intermediate3-(4-(2-aminoethylamino)-1,2,5-oxadiazol-3-yl)-4-(3-bromo-4-fluorophenyl)-1,2,4-oxadiazol-5(4H)-onehydroiodide

Step 1:4-Amino-N′-hydroxy-N-(2-methoxyethyl)-1,2,5-oxadiazole-3-carboximidamide

4-Amino-N-hydroxy-1,2,5-oxadiazole-3-carboximidoyl chloride (can beprepared according to Example 5, steps 1-2, 200.0 g, 1.23 mol) was mixedwith ethyl acetate (1.2 L). At 0-5° C. 2-methoxyethylamine [Aldrich,product #143693] (119.0 mL, 1.35 mol) was added in one portion whilestirring. The reaction temperature rose to 41° C. The reaction wascooled to 0-5° C. Triethylamine (258 mL, 1.84 mol) was added. Afterstirring 5 min, LCMS indicated reaction completion. The reactionsolution was washed with water (500 mL) and brine (500 mL), dried oversodium sulfate, and concentrated to give the desired product (294 g,119%) as a crude dark oil. LCMS for C₆H₁₂N₅O₃ (M+H)⁺: m/z=202.3. ¹H NMR(400 MHz, DMSO-d₆): δ 10.65 (s, 1H), 6.27 (s, 2H), 6.10 (t, J=6.5 Hz,1H), 3.50 (m, 2H), 3.35 (d, J=5.8 Hz, 2H), 3.08 (s, 3H).

Step 2:1V-Hydroxy-4-[(2-methoxyethyl)amino]-1,2,5-oxadiazole-3-carboximidamide

4-Amino-N′-hydroxy-N-(2-methoxyethyl)-1,2,5-oxadiazole-3-carboximidamide(248.0 g, 1.23 mol) was mixed with water (1 L). Potassium hydroxide (210g, 3.7 mol) was added. The reaction was refluxed at 100° C. overnight(15 hours). TLC with 50% ethyl acetate (containing 1% ammoniumhydroxide) in hexane indicated reaction completed (product Rf=0.6,starting material Rf=0.5). LCMS also indicated reaction completion. Thereaction was cooled to room temperature and extracted with ethyl acetate(3×1 L). The combined ethyl acetate solution was dried over sodiumsulfate and concentrated to give the desired product (201 g, 81%) as acrude off-white solid. LCMS for C₆H₁₂N₅O₃ (M+H)⁺: m/z=202.3 ¹H NMR (400MHz, DMSO-d₆): δ 10.54 (s, 1H), 6.22 (s, 2H), 6.15 (t, J=5.8 Hz, 1H),3.45 (t, J=5.3 Hz, 2H), 3.35 (m, 2H), 3.22 (s, 3H).

Step 3:N-Hydroxy-4-[(2-methoxyethyl)amino]-1,2,5-oxadiazole-3-carboximidoylchloride

At room temperatureN′-hydroxy-4-[(2-methoxyethyl)amino]-1,2,5-oxadiazole-3-carboximidamide(50.0 g, 0.226 mol) was dissolved in 6.0 M hydrochloric acid aqueoussolution (250 mL, 1.5 mol). Sodium chloride (39.5 g, 0.676 mol) wasadded followed by water (250 mL) and ethyl acetate (250 mL). At 3-5° C.a previously prepared aqueous solution (100 mL) of sodium nitrite (15.0g, 0.217 mol) was added slowly over 1 hr. The reaction was stirred at3-8° C. for 2 hours and then room temperature over the weekend. LCMSindicated reaction completed. The reaction solution was extracted withethyl acetate (2×200 mL). The combined ethyl acetate solution was driedover sodium sulfate and concentrated to give the desired product (49.9g, 126%) as a crude white solid. LCMS for C₆H₁₀ClN₄O₃ (M+H)⁺: m/z=221.0.¹H NMR (400 MHz, DMSO-d₆): δ 13.43 (s, 1H), 5.85 (t, J=5.6 Hz, 1H), 3.50(t, J=5.6 Hz, 2H), 3.37 (dd, J=10.8, 5.6 Hz, 2H), 3.25 (s, 3H).

Step 4:N-(3-Bromo-4-fluorophenyl)-N′-hydroxy-4-[(2-methoxyethyl)amino]-1,2,5-oxadiazole-3-carboximidamide

N-Hydroxy-4-[(2-methoxyethyl)amino]-1,2,5-oxadiazole-3-carboximidoylchloride (46.0 g, 0.208 mol) was mixed with water (300 mL). The mixturewas heated to 60° C. 3-Bromo-4-fluoroaniline [Oakwood products, product#013091] (43.6 g, 0.229 mol) was added and stirred for 10 min. A warmsodium bicarbonate (26.3 g, 0.313 mol) solution (300 mL water) was addedover 15 min. The reaction was stirred at 60° C. for 20 min. LCMSindicated reaction completion. The reaction solution was cooled to roomtemperature and extracted with ethyl acetate (2×300 mL). The combinedethyl acetate solution was dried over sodium sulfate and concentrated togive the desired product (76.7 g, 98%) as a crude brown solid. LCMS forC₁₂H₁₄BrFN₅O₃ (M+H)⁺: m/z=374.0, 376.0. ¹H NMR (400 MHz, DMSO-d₆): δ11.55 (s, 1H), 8.85 (s, 1H), 7.16 (t, J=8.8 Hz, 1H), 7.08 (dd, J=6.1,2.7 Hz, 1H), 6.75 (m, 1H), 6.14 (t, J=5.8 Hz, 1H), 3.48 (t, J=5.2 Hz,2H), 3.35 (dd, J=10.8, 5.6 Hz, 2H), 3.22 (s, 3H).

Step 5:4-(3-Bromo-4-fluorophenyl)-3-{4-[(2-methoxyethyl)amino]-1,2,5-oxadiazol-3-yl}-1,2,4-oxadiazol-5(4H)-one

A mixture ofN-(3-bromo-4-fluorophenyl)-N′-hydroxy-4-[(2-methoxyethyl)amino]-1,2,5-oxadiazole-3-carboximidamide(76.5 g, 0.204 mol), 1,1′-carbonyldiimidazole (49.7 g, 0.307 mol), andethyl acetate (720 mL) was heated to 60° C. and stirred for 20 min. LCMSindicated reaction completed. The reaction was cooled to roomtemperature, washed with 1 N HCl (2×750 mL), dried over sodium sulfate,and concentrated to give the desired product (80.4 g, 98%) as a crudebrown solid. LCMS for C₁₃H₁₂BrFN₅O₄ (M+H)⁺: m/z=400.0, 402.0. ¹H NMR(400 MHz, DMSO-d₆): δ 7.94 (t, J=8.2 Hz, 1H), 7.72 (dd, J=9.1, 2.3 Hz,1H), 7.42 (m, 1H), 6.42 (t, J=5.7 Hz, 1H), 3.46 (t, J=5.4 Hz, 2H), 3.36(t, J=5.8 Hz, 2H), 3.26 (s, 3H).

Step 6:4-(3-Bromo-4-fluorophenyl)-3-{4-[(2-hydroxyethyl)amino]-1,2,5-oxadiazol-3-yl}-1,2,4-oxadiazol-5(4H)-one

4-(3-Bromo-4-fluorophenyl)-3-{4-[(2-methoxyethyl)amino]-1,2,5-oxadiazol-3-yl}-1,2,4-oxadiazol-5(4H)-one(78.4 g, 0.196 mol) was dissolved in dichloromethane (600 mL). At −67°C. boron tribromide (37 mL, 0.392 mol) was added over 15 min. Thereaction was warmed up to −10° C. in 30 min. LCMS indicated reactioncompleted. The reaction was stirred at room temperature for 1 hour. At0-5° C. the reaction was slowly quenched with saturated sodiumbicarbonate solution (1.5 L) over 30 min. The reaction temperature roseto 25° C. The reaction was extracted with ethyl acetate (2×500 mL, firstextraction organic layer is on the bottom and second extraction organiclager is on the top). The combined organic layers were dried over sodiumsulfate and concentrated to give the desired product (75 g, 99%) as acrude brown solid. LCMS for C₁₂H₁₀BrFN₅O₄ (M+H)⁺: m/z=386.0, 388.0. ¹HNMR (400 MHz, DMSO-d₆): δ 8.08 (dd, J=6.2, 2.5 Hz, 1H), 7.70 (m, 1H),7.68 (t, J=8.7 Hz, 1H), 6.33 (t, J=5.6 Hz, 1H), 4.85 (t, J=5.0 Hz, 1H),3.56 (dd, J=10.6, 5.6 Hz, 2H), 3.29 (dd, J=11.5, 5.9 Hz, 2H).

Step 7:2-({4-[4-(3-Bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}amino)ethylmethanesulfonate

4-(3-bromo-4-fluorophenyl)-3-(4-(2-hydroxyethylamino)-1,2,5-oxadiazol-3-yl)-1,2,4-oxadiazol-5(4H)-one(72.2 g, 0.188 mol) was mixed with ethyl acetate (600 mL).Methanesulfonyl chloride (19.2 mL, 0.248 mol) was added followed bytriethylamine (34.9 mL, 0.250 mol). The reaction was stirred at roomtemperature for 5 min. When LCMS indicated completion of reaction(M+H=442), 500 mL of water was added into reaction. The reaction wasextracted with ethyl acetate (2×500 mL). The combined ethyl acetatesolution was washed with brine (500 mL), dried over sodium sulfate andconcentrated to give 85.1 g crude brown solid. ¹H NMR verified thestructure. Crude yield was 97%. LCMS for C₁₃H₁₁BrFN₅O₆SNa (M+Na)⁺:m/z=485.9, 487.9. ¹H NMR (400 MHz, DMSO-d₆): δ 8.08 (dd, J=6.2, 2.5 Hz,1H), 7.72 (m, 1H), 7.58 (t, J=8.7 Hz, 1H), 6.75 (t, J=5.9 Hz, 1H), 4.36(t, J=5.3 Hz, 2H), 3.58 (dd, J=11.2, 5.6 Hz, 2H), 3.18 (s, 3H).

Step 8:3-{4-[(2-Azidoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-(3-bromo-4-fluorophenyl)-1,2,4-oxadiazol-5(4H)-one

2-(4-(4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)-1,2,5-oxadiazol-3-ylamino)ethylmethanesulfonate (50.0 g, 0.108 mol) was dissolved in N,N-dimethylformamide (83 mL). Sodium azide (10.5 g, 0.162 mol) was added.The reaction was stirred at 65° C. for 5-6 hours. LCMS indicatedreaction completed (M+Na=435). The reaction was quenched with water (250mL) and extracted with ethyl acetate (2×250 mL). The combined ethylacetate solution was washed with water (250 mL, layer separation wasslow, 100 mL of brine was added to improve the separation), dried oversodium sulfate, and concentrated to give 49.7 g crude brown solid. Crudeyield is 112%. LCMS for C₁₂H₈BrFN₈O₃Na (M+Na)⁺: m/z=433.0, 435.0. ¹H NMR(400 MHz, DMSO-d₆): δ 8.08 (dd, J=6.2, 2.5 Hz, 1H), 7.72 (m, 1H), 7.58(t, J=8.7 Hz, 1H), 6.75 (t, J=5.7 Hz, 1H), 3.54 (t, J=5.3 Hz, 2H), 3.45(dd, J=11.1, 5.2 Hz, 2H).

Step 9:3-(4-(2-aminoethylamino)-1,2,5-oxadiazol-3-yl)-4-(3-bromo-4-fluorophenyl)-1,2,4-oxadiazol-5(4H)-onehydroiodide

3-(4-(2-azidoethylamino)-1,2,5-oxadiazol-3-yl)-4-(3-bromo-4-fluorophenyl)-1,2,4-oxadiazol-5(4H)-one(80.0 g, 0.194 mol) was mixed with methanol (800 mL). Sodium iodide(175.0 g, 1.17 mol) was added. The reaction was stirred at roomtemperature for 10 min. Chlorotrimethylsilane (148 mL, 1.17 mol) wasdissolved in methanol (100 mL) and added to the reaction over 30 min.The reaction temperature rose 42° C. The reaction was stirred at roomtemperature for 30 min. LCMS indicated reaction completed (M+H=386). Thereaction was quenched with sodium thiosulfate (190.0 g, 1.20 mol) inwater (900 mL). A large amount of solid precipitated. The product wascollected by filtration (filtration speed was slow), rinsed with water(200 mL), and dried on vacuum overnight. The filter cake was slurried inethyl acetate (500 mL) for 30 min. The product was filtered (filtrationspeed is slow) and dried under vacuum over weekend to give 95 g of anoff-white solid. LCMS for C₁₂H₁₁BrFN₆O₃ (M+H)⁺: m/z=384.9, 386.9. ¹H NMR(400 MHz, DMSO-d₆): δ 8.12 (m, 4H), 7.76 (m, 1H), 7.58 (t, J=8.7 Hz,1H), 6.78 (t, J=6.1 Hz, 1H), 3.51 (dd, J=11.8, 6.1 Hz, 2H), 3.02 (m,2H).

Example 23 Alternate preparation of4-({2-[(Aminosulfonyl)amino]ethyl}amino)-N-(3-bromo-4-fluorophenyl)-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide

Step 1:4-(3-bromo-4-fluorophenyl)-3-(4-(2-hydroxyethylamino)-1,2,5-oxadiazol-3-yl)-1,2,4-oxadiazol-5(4H)-one

To a solution of4-(3-bromo-4-fluorophenyl)-3-(4-(2-methoxyethylamino)-1,2,5-oxadiazol-3-yl)-1,2,4-oxadiazol-5(4H)-one(can be prepared according to Example 5, steps 1-7; 1232 g, 3.08 mol) indichloromethane (12 L) stirring in a 22 L flask at 0° C. was added borontribromide (354 mL, 3.67 mL) dropwise at a rate so that the temperaturedid not exceed 10° C. After stirring on ice for 1 h, a solution ofsaturated aqueous sodium bicarbonate (2 L) was carefully added at a rateso that the temperature did not exceed 20° C. (addition time 10 min).The resulting mixture was transferred to a 50 L separatory funnel,diluted with water (10 L), and the pH of the aqueous layer adjusted from1 to 8 using solid sodium bicarbonate. The layers were separated, andthe organic layer was washed with water (10 L), and the solvents removedin vacuo to afford a tan solid (24 mol processed in multiple runs, 9.54kg, quant. yield). The material was slurried in 4 volumes of 7:1heptane:ethyl acetate (4×22 L flasks), filtered, and dried to furnishthe title compound as a tan solid (8679 g, 94%). The product was amixture of the hydroxy- and the corresponding bromo-species.

Step 2:2-(4-(4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)-1,2,5-oxadiazol-3-ylamino)ethylmethanesulfonate

To a solution of4-(3-bromo-4-fluorophenyl)-3-{4-[(2-hydroxyethyl)amino]-1,2,5-oxadiazol-3-yl}-1,2,4-oxadiazol-5(4H)-one(1.5 kg, 3.9 mol, containing also some of the correspondingbromo-compound) in ethyl acetate (12 L) was added methanesulfonylchloride (185 mL, 2.4 mol) dropwise over 1 h at room temperature.Triethylamine (325 mL, 2.3 mol) was added dropwise over 45 min, duringwhich time the reaction temperature increased to 35° C. After 2 h, thereaction mixture was washed with water (5 L), brine (1 L), dried oversodium sulfate, combined with 3 more reactions of the same size, and thesolvents removed in vacuo to afford the desired product (7600 g,quantitative yield, containing also some of the correspondingbromo-compound, Caution: irritating dust!) as a tan solid. LCMS forC₁₃H₁₁BrFN₅O₆SNa (M+Na)⁺: m/z=485.9, 487.9. ¹H NMR (400 MHz, DMSO-d₆): δ8.08 (dd, J=6.2, 2.5 Hz, 1H), 7.72 (m, 1H), 7.58 (t, J=8.7 Hz, 1H), 6.75(t, J=5.9 Hz, 1H), 4.36 (t, J=5.3 Hz, 2H), 3.58 (dd, J=11.2, 5.6 Hz,2H), 3.18 (s, 3H).

Step 3:3-(4-(2-azidoethylamino)-1,2,5-oxadiazol-3-yl)-4-(3-bromo-4-fluorophenyl)-1,2,4-oxadiazol-5(4H)-one

To a solution of2-({4-[4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}amino)ethylmethanesulfonate (2.13 kg, 4.6 mol, containing also some of thecorresponding bromo-compound) in dimethylformamide (4 L) stirring in a22 L flask was added sodium azide (380 g, 5.84 mol). The reaction washeated at 50° C. for 6 h, poured into ice/water (8 L), and extractedwith 1:1 ethyl acetate:heptane (20 L). The organic layer was washed withwater (5 L) and brine (5 L), and the solvents removed in vacuo to affordthe desired product (1464 g, 77%) as a tan solid. LCMS forC₁₂H₈BrFN₈O₃Na (M+Na)⁺: m/z=433.0, 435.0. ¹H NMR (DMSO-d₆, 400 MHz): δ8.08 (dd, J=6.2, 2.5 Hz, 1H), 7.72 (m, 1H), 7.58 (t, J=8.7 Hz, 1H), 6.75(t, J=5.7 Hz, 1H), 3.54 (t, J=5.3 Hz, 2H), 3.45 (dd, J=11.1, 5.2 Hz,2H).

Step 4:3-{4-[(2-Aminoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-(3-bromo-4-fluorophenyl)-1,2,4-oxadiazol-5(4H)-onehydrochloride

Step 4, Part 1: tert-Butyl2-(4-(4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)-1,2,5-oxadiazol-3-ylamino)ethylcarbamate

Sodium iodide (1080 g, 7.2 mol) was added to3-{4-[(2-azidoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-(3-bromo-4-fluorophenyl)-1,2,4-oxadiazol-5(4H)-one(500 g, 1.22 mol) in methanol (6 L). The mixture was allowed to stir for30 min during which time a mild exotherm was observed.Chlorotrimethylsilane (930 mL, 7.33 mol) was added as a solution inmethanol (1 L) dropwise at a rate so that the temperature did not exceed35° C., and the reaction was allowed to stir for 3.5 h at ambienttemperature. The reaction was neutralized with 33 wt % solution ofsodium thiosulfate pentahydrate in water (˜1.5 L), diluted with water (4L), and the pH adjusted to 9 carefully with solid potassium carbonate(250 g added in small portions: watch foaming). Di-tert-butyldicarbonate (318 g, 1.45 mol) was added and the reaction was allowed tostir at room temperature. Additional potassium carbonate (200 g) wasadded in 50 g portions over 4 h to ensure that the pH was still at orabove 9. After stirring at room temperature overnight, the solid wasfiltered, triturated with water (2 L), and then MTBE (1.5 L). A total of11 runs were performed (5.5 kg, 13.38 mol). The combined solids weretriturated with 1:1 THF:dichloromethane (24 L, 4 runs in a 20 L rotaryevaporator flask, 50° C., 1 h), filtered, and washed withdichloromethane (3 L each run) to afford an off-white solid. The crudematerial was dissolved at 55° C. tetrahydrofuran (5 mL/g), treated withdecolorizing carbon (2 wt %) and silica gel (2 wt %), and filtered hotthrough celite to afford the product as an off-white solid (5122 g). Thecombined MTBE, THF, and dichloromethane filtrates were concentrated invacuo and chromatographed (2 kg silica gel, heptane with a 0-100% ethylacetate gradient, 30 L) to afford more product (262 g). The combinedsolids of tert-butyl2-(4-(4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)-1,2,5-oxadiazol-3-ylamino)ethylcarbamatewere dried to a constant weight in a convection oven (5385 g, 83%).

Step 5, Part 2:3-{4-[(2-Aminoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-(3-bromo-4-fluorophenyl)-1,2,4-oxadiazol-5(4H)-onehydrochloride

Method A:

In a 22 L flask was charged hydrogen chloride (4 N solution in1,4-dioxane, 4 L, 16 mol). tert-Butyl[2-({4-[4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}amino)ethyl]carbamate(2315 g, 4.77 mol) was added as a solid in portions over 10 min. Theslurry was stirred at room temperature and gradually became a thickpaste that could not be stirred. After sitting overnight at roomtemperature, the paste was slurried in ethyl acetate (10 L), filtered,re-slurried in ethyl acetate (5 L), filtered, and dried to a constantweight to afford the desired product as a white solid (combined withother runs, 5 kg starting material charged, 4113 g, 95%). LCMS forC₁₂H₁₁BrFN₆O₃ (M+H)⁺: m/z=384.9, 386.9. ¹H NMR (400 MHz, DMSO-d₆): δ8.12 (m, 4H), 7.76 (m, 1H), 7.58 (t, J=8.7 Hz, 1H), 6.78 (t, J=6.1 Hz,1H), 3.51 (dd, J=11.8, 6.1 Hz, 2H), 3.02 (m, 2H).

Method B:

tert-Butyl[2-({4-[4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}amino)ethyl]carbamate(5000 g) was added to a mixture of isopropanol (20 L) and 4 N HCl in1,4-dioxane (10 L) at room temperature. The batch was heated to 40-45°C. and held for 1 h. Ethyl acetate was added to the batch at 40-45° C.and held for 2.5 h. Upon reaction completion, as indicated by HPLC,heptane (10 L) was added to the batch. The batch was cooled to 25° C.The product was isolated by filtration and the wet cake was washed withethyl acetate (3×5.0 L). The product was dried in a vacuum, oven at 20°C. to give 4344 g (93.4% yield) of the title compound. LC-MS, ¹H and ¹³CNMR, and HPLC data of this lot were identical to those of the productprepared by Method A.

Step 5: tert-Butyl({[2-({4-[4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}amino)ethyl]amino}sulfonyl)carbamate

A 5 L round bottom flask was charged with chlorosulfonyl isocyanate[Aldrich, product #142662] (149 mL, 1.72 mol) and dichloromethane (1.5L) and cooled using an ice bath to 2° C. tert-Butanol (162 mL, 1.73 mol)in dichloromethane (200 mL) was added dropwise at a rate so that thetemperature did not exceed 10° C. The resulting solution was stirred atroom temperature for 30-60 min to provide tert-butyl[chlorosulfonyl]carbamate.

A 22 L flask was charged with3-{4-[(2-aminoethyl)amino]-1,2,5-oxadiazol-3-yl}-4-(3-bromo-4-fluorophenyl)-1,2,4-oxadiazol-5(4H)-onehydrochloride (661 g, 1.57 mol) and 8.5 L dichloromethane. After coolingto −15° C. with an ice/salt bath, the solution of tert-butyl[chlorosulfonyl]carbamate (prepared as above) was added at a rate sothat the temperature did not exceed −10° C. (addition time 7 min). Afterstirring for 10 min, triethylamine (1085 mL, 7.78 mol) was added at arate so that the temperature did not exceed −5° C. (addition time 10min). The cold bath was removed, the reaction was allowed to warm to 10°C., split into two portions, and neutralized with 10% conc HCl (4.5 Leach portion). Each portion was transferred to a 50 L separatory funneland diluted with ethyl acetate to completely dissolve the white solid(˜25 L). The layers were separated, and the organic layer was washedwith water (5 L), brine (5 L), and the solvents removed in vacuo toafford an off-white solid. The solid was triturated with MTBE (2×1.5 L)and dried to a constant weight to afford a white solid. A total of 4113g starting material was processed in this manner (5409 g, 98%). ¹H NMR(400 MHz, DMSO-d₆): δ 10.90 (s, 1H), 8.08 (dd, J=6.2, 2.5 Hz, 1H), 7.72(m, 1H), 7.59 (t, J=8.6 Hz, 1H), 6.58 (t, J=5.7 Hz, 1H), 3.38 (dd,J=12.7, 6.2 Hz, 2H), 3.10 (dd, J=12.1, 5.9 Hz, 2H), 1.41 (s, 9H).

Step 6:N-[2-({4-[4-(3-Bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}amino)ethyl]sulfamide

Method A: Using Trifluoroacetic Acid

To a 22 L flask containing 98:2 trifluoroacetic acid:water (8.9 L) wasadded tert-butyl({[2-({4-[4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}amino)ethyl]amino}sulfonyl)carbamate(1931 g, 3.42 mol) in portions over 10 minutes. The resulting mixturewas stirred at room temperature for 1.5 h, the solvents removed invacuo, and chased with dichloromethane (2 L). The resulting solid wastreated a second time with fresh 98:2 trifluoroacetic acid:water (8.9L), heated for 1 h at 40-50° C., the solvents removed in vacuo, andchased with dichloromethane (3×2 L). The resulting white solid was driedin a vacuum drying oven at 50° C. overnight. A total of 5409 g wasprocessed in this manner (4990 g, quant. yield). LCMS for C₁₂H₁₂BrFN₇O₅S(M+H)⁺: m/z=463.9, 465.9. ¹H NMR (400 MHz, DMSO-d₆): δ 8.08 (dd, J=6.2,2.5 Hz, 1H), 7.72 (m, 1H), 7.59 (t, J=8.7 Hz, 1H), 6.67 (t, J=5.9 Hz,1H), 6.52 (t, J=6.0 Hz, 1H), 3.38 (dd, J=12.7, 6.3 Hz, 2H), 3.11 (dd,J=12.3, 6.3 Hz).

Method B: Using Hydrochloric Acid

To solution of tert-butyl({[2-({4-[4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}amino)ethyl]amino}sulfonyl)carbamate(4500 g) in isopropanol (9 L) was added 4 N HCl in dioxane (8.0 L). Thereaction mixture was heated to 40-45° C. and was held at thistemperature for about 5 h. Upon completion of reaction (as indicated byHPLC analysis), heptane (72 L) was added to the reaction mixture. Theresultant mixture was heated to 68° C. and held at this temperature for1 h. The batch was allowed to cool to about 23° C. The solid product wascollected by filtration. The wet cake was washed with a mixture ofheptane (16 L) and isopropanol (1.2 L) and dried under suction on afilter funnel. The crude product was dissolved in ethyl acetate (10.8 L)at about 43° C. Heptane (32.4 L) was added to the ethyl acetate solutionover 15 min. The batch was heated to 70° C. and held at this temperaturefor 1 h. The batch was cooled to 21° C. and solid product was collectedby filtration. The wet cake was washed with heptane (14.4 L) and driedunder suction on the filter funnel. Yield of product was 3034 g. LC-MS,¹H and ¹³C NMR, and HPLC data of this lot were identical to those of theproduct prepared by Method A.

Step 7:(Z)-4-({2-[(Aminosulfonyl)amino]ethyl}amino)-N-(3-bromo-4-fluorophenyl)-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide

Method A:

To a crude mixture ofN-[2-({4-[4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}amino)ethyl]sulfamide(2.4 mol) containing residual amounts of trifluoroacetic acid stirringin a 22 L flask was added THF (5 L). The resulting solution was cooledto 0° C. using an ice bath and 2 N NaOH (4 L) was added at a rate sothat the temperature did not exceed 10° C. After stirring at ambienttemperature for 3 h (LCMS indicated no starting material remained), thepH was adjusted to 3-4 with concentrated HCl (˜500 mL). The THF wasremoved in vacuo, and the resulting mixture was extracted with ethylacetate (15 L). The organic layer was washed with water (5 L), brine (5L), and the solvents removed in vacuo to afford a solid. The solid wastriturated with MTBE (2×2 L), combined with three other reactions of thesame size, and dried overnight in a convection oven to afford a whitesolid (3535 g). The solid was recrystallized (3×22 L flasks, 2:1deionized ultra-filtered water:ethanol, 14.1 L each flask) and dried ina 50° C. convection oven to a constant weight to furnish the titlecompound as an off-white solid (3290 g, 78%). LCMS for C₁₁H₁₄BrFN₇O₄S(M+H)⁺: m/z=437.9, 439.9. ¹H NMR (400 MHz, DMSO-d₆): δ 11.51 (s, 1H),8.90 (s, 1H), 7.17 (t, J=8.8 Hz, 1H), 7.11 (dd, J=6.1, 2.7 Hz, 1H), 6.76(m, 1H), 6.71 (t, J=6.0 Hz, 1H), 6.59 (s, 2H), 6.23 (t, J=6.1 Hz, 1H),3.35 (dd, J=10.9, 7.0 Hz, 2H), 3.10 (dd, J=12.1, 6.2 Hz, 2H). X-raycrystallographic analysis determined that the title compound adopts aZ-configuration (Z-isomer) with respect to the carbon-nitrogen doublebond (C═N) of oxime functionality.

Method B:

N-[2-({4-[4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl]-1,2,5-oxadiazol-3-yl}amino)ethyl]sulfamide(1500 g) was added to THF (6.0 L) and the batch was cooled to 2° C.Trifluoroacetic acid (0.006 L) was added to the batch at 2° C. followedby addition of aqueous sodium hydroxide solution (384 g of solid NaOH in4.8 L of water) at 0-2° C. The batch was warmed up to about 16° C. andheld for 5 h. Upon completion of reaction, as indicated by HPLC,concentrated hydrochloric acid (0.7 L) was added to adjust the pH of thebatch to 3-4. About 4 L of solvent was removed from the batch bydistillation under reduced pressure. The batch was added to ethylacetate (18.0 L) and the biphasic mixture was stirred for 15 min. Theorganic layer was washed with water (6.0 L) and brine (6.0 L)sequentially. The organic solution was dried over anhydrous magnesiumsulfate. Magnesium sulfate was filtered and the filtrate was evaporatedto dryness under reduced pressure. To the resultant solid, MTBE (3.0 L)was added and the slurry was stirred for 15 min. The solid product wasisolated by filtration. The filter cake was washed with MTBE (1.2 L) andheptane (1.2 L) sequentially. The solid was dried on the filter funnelunder suction to give 1416 g (87.9%) of the product. The product (2440g, obtained in two batches) was further purified by re-slurrying in MTBE(9.6 L) at 17° C. for 2 h. The batch was cooled to 6° C. for 30 min. Thesolid product was collected by filtration and the wet cake was washedwith MTBE (3.6 L) and heptane (1.2 L) sequentially. The product wasdried in a vacuum oven at 20° C. to give 1962 g of the title compound in81.7% yield. LC-MS, ¹H and ¹³C NMR, and HPLC data of this lot wereidentical to those of the product prepared by Method A.

Example 244-(2-[(Aminosulfonyl)(methyl)amino]ethylamino)-N-(3-chloro-4-fluorophenyl)-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide

Step 1. (4-Methoxybenzyl)methylamine

40% Aqueous methylamine (160 mL, 2.40 mol) was added to a solution of4-methoxybenzaldehyde (164 g, 1.20 mol) in MeOH (600 mL). The mixturewas diluted with additional MeOH (600 mL) and placed in a water bath.NaBH₄ (24.0 g, 0.63 mol) was added in 1 g portions over 2 h. Ice wasadded periodically to the water bath to maintain the reactiontemperature at 15-20° C. throughout the addition of NaBH₄. The reactionmixture was stirred 0.5. The mixture was cooled to 5° C. and adjusted topH 1 by the cautious addition of 6N HCl (680 mL). MeOH was removed underreduced pressure and the aqueous residue was extracted with EtOAc (500mL). The EtOAc phase was back extracted with 1N HCl (200 mL). Thecombined acidic solution was cooled in an ice-bath and made basic (pH11-12) with 24% w/v NaOH until all solids that initially form dissolved.The aqueous mixture was extracted with EtOAc (4×400 mL). The combinedEtOAc extracts were dried over Na₂SO₄, filtered and the solutionconcentrated under reduced pressure. The residual liquid was distilledto give 150 g of (4-methoxybenzyl)methylamine (bp 79-88°, 1.0 Torr).

Step 2. [(4-Methoxybenzyl)methylamino]acetonitrile

A solution of (4-methoxybenzyl)methylamine (125 g, 0.828 mol),chloroacetonitrile (75 g, 0.993 mol, 1.20 equiv) andN,N-diisopropylethylamine (134 g, 1.035 mol) in toluene (600 mL) wasrefluxed for 1 h. The mixture was cooled to room temperature and dilutedwith EtOAc (1.5 L). The solution was washed with H₂O (3×750 mL), brine,dried over Na₂SO₄, filtered and the solution concentrated under reducedpressure. The residual liquid was distilled to give 152 g (97%) of[(4-methoxybenzyl)methylamino]acetonitrile (bp 128-135°, 2.0 Torr).

Step 3. N¹-4-methoxybenzyl-N¹-methylethanediamine

1M LiAlH₄ in THF (1.10 L, 1.10 mol) was added to THF (1 L) and thesolution cooled to −20° C. A solution of[(4-methoxybenzyl)methylamino]acetonitrile (140 g, 0.737 mol) in THF(500 mL) was added over 1 h while maintaining the reaction temperatureat <−10° C. The mixture was allowed to warm to room temperature andstirred 1 h. The mixture was cooled to −10° C. and a solution of sodiumpotassium tartrate (37% w/v, 200 mL) was added dropwise cautiously whilemaintaining reaction temperature at <0° C. Celite (50 g) was added andthe mixture filtered through a pad of Celite, washing the solids withTHF (300 mL) followed by MTBE (300 mL). The filtrate was dried overNa₂SO₄, filtered and the solution concentrated under reduced pressure.The residual liquid was Kugelrohr distilled to give 114 g (80%) ofN¹-4-methoxybenzyl-N¹-methylethanediamine (oven temperature 128-135°,1.0 Torr).

Step 4.4-Amino-N′-hydroxy-N-{2-[(4-methoxybenzyl)methyl)amino]ethyl}-1,2-5-oxadiazole-3-carboximidamide

A solution of N′-4-methoxybenzyl-M-methylethanediamine (17.7 g, 0.109mol) in THF (50 mL) and EtOAc (200 mL) was added over 0.5 h at >25° C.(mild cooling required) to a solution of 5 (21.2 g, 0.109 mol) and Et₃N(12.7 g, 17.5 ml, 0.126 mol) in EtOAc (500 mL). The pale yellowsuspension was stirred at room temperature for 1 hr. The mixture wasfiltered and the solids were washed with EtOAc (200 mL). The filtratewas washed with H₂O (3×400 mL), brine (400 mL), dried over Na₂SO₄,filtered and the solution concentrated under reduced pressure to ˜300 mLvolume. Heptane (300 mL) was added and the solution concentrated underreduced pressure until a slight turbidity developed. The mixture wasstirred until crystallization was complete (˜1 h) and then concentratedto near dryness. The solid was triturated with heptane, filtered anddried to give 32.6 g (93%) of4-amino-N′-hydroxy-N-{2-[(4-methoxybenzyl)methyl)amino]ethyl}-1,2-5-oxadiazole-3-carboximidamideas an off-white solid.

Step 5.N′-hydroxy-4-({2-[(4-methoxybenzyl)(methyl)amino]ethyl}amino)-1,2-5-oxadiazole-3-carboximidamide

KOH (34.0 g, 0.607 mol) was added to a suspension of4-amino-N′-hydroxy-N-{2-[(4-methoxybenzyl)methyl)amino]ethyl}-1,2-5-oxadiazole-3-carboximidamide(77.7 g, 0.243 mol) in ethylene glycol (500 mL) and the mixture heatedto ˜135° C. After 2 h at ˜135° C. the mixture was cooled to roomtemperature and stirred overnight. The mixture was diluted with H₂O (1.5L) and extracted with MTBE (3×750 mL, 2×500 mL). The combined organicsolution was washed with H₂O (4×750 mL), brine (750 mL), dried overNa₂SO₄, filtered and the solution concentrated under reduced pressure tonear dryness. The red-maroon viscous oil was dissolved in 50%MTBE/heptane (500 mL) with slight warming, seeded and the mixturestirred 0.5 h. The suspension was concentrated to ˜½ volume, filteredand the solids washed with 10% MTBE/heptane (350 mL) and dried to give68.1 g (88%) of N′-hydroxy-4-({2-[(4-methoxybenzyl)(methyl)amino]ethyl}amino)-1,2-5-oxadiazole-3-carboximidamide as a light pinkish-whitesolid.

Step 6.N-Hydroxy-4-(2-[(4-methoxybenzyl)(methyl)amino]ethylamino)-1,2-5-oxadiazole-3-carboximidoylchloride hydrochloride

A mixture ofN′-hydroxy-4-({2-[(4-methoxybenzyl)(methyl)amino]ethyl}amino)-1,2-5-oxadiazole-3-carboximidamide(32.4 g, 0.101 mol), NaCl (17.6 g, 0.304 mol), HOAc (175 mL) and 6 N HCl(86 mL) was stirred at room temperature until all solids (except someNaCl) had dissolved and then cooled in an ice/brine bath. A solution ofNaNO₂(7.7 g, 0.111 mol) in H₂O (40 mL) was added dropwise maintainingthe reaction temperature at −2 to 2° C. When addition was complete themixture was stirred at 0-3° with an extremely thick suspension formingafter 0.75 h. After 2 h the mixture was allowed to warm to roomtemperature and then it was concentrated under reduced pressure to givea pale yellow solid. Residual HOAc and H₂O were co-evaporated withtoluene (3×400 mL) and the solid was dried under high vacuum overnight.The crude mixture of N-hydroxy-4-(2-[(4-methoxybenzyl)(methyl)amino]ethyl amino)-1,2-5-oxadiazole-3-carboximidoyl chloride hydrochloride andNaCl was used in the next reaction without further purification.

Step 7.N-(3-Chloro-4-fluorophenyl)-N′-hydroxy-4-(2-[(4-methoxybenzyl)-(methyl)amino]ethylamino)-1,2-5-oxadiazole-3-carboximidamide

Crude N-Hydroxy-4-(2-[(4-methoxybenzyl)(methyl)amino] ethylamino)-1,2-5-oxadiazole-3-carboximidoyl chloride hydrochloride wassuspended in EtOH (650 mL) and 3-chloro-4-fluoroaniline (12) (36.7 g,0.253 mol) was added. The mixture was refluxed for 5 h and then cooledto room temperature. The mixture was concentrated under reduced pressureto remove EtOH. The residual oily solid was partitioned between EtOAc (1L) and ½ saturated aqueous NaHCO₃ solution (300 mL). The organic phasewas washed with brine (300 mL), dried over Na₂SO₄, filtered and thesolution concentrated under reduced pressure. The resulting oily solidwas triturated with 30% MTBE/heptane (700 mL), filtered, washed with 30%MTBE/heptane (300 mL) and dried to give 37.9 g (83% fromN′-hydroxy-4-({2-[(4-methoxybenzyl)(methyl)amino]ethyl}amino)-1,2-5-oxadiazole-3-carboximidamide) ofN-(3-chloro-4-fluorophenyl)-N′-hydroxy-4-(2-[(4-methoxybenzyl)-(methyl)amino]ethyl amino)-1,2-5-oxadiazole-3-carboximidamide as a grayish-tan solid.

Step 8.4-(3-Chloro-4-fluorophenyl)-3-[4-(2-[(4-methoxybenzyl)-(methyl)amino]-ethylamino)-1,2-5-oxadiazol-3-yl]-1,2,4-oxadiazol-5(4H)-one

N,N-Carbonyldiimidazole (16.4 g, 101 mmol) was added to a solution ofN-(3-chloro-4-fluorophenyl)-N′-hydroxy-4-(2-[(4-methoxybenzyl)-(methyl)amino]ethyl amino)-1,2-5-oxadiazole-3-carboximidamide (37.9 g, 84.4 mmol) inTHF (550 mL) and the mixture refluxed 1.75 h. The reaction mixture wascooled to room temperature and concentrated under reduced pressure. Theresulting brown solid was dissolved in EtOAc (1 L) and the solutionwashed with H₂O (3×500 mL), brine (300 mL), dried over Na₂SO₄, filteredand the solution concentrated under reduced pressure to 300 mL. Heptane(400 mL) was added and the mixture was concentrated to a thick slurry.The slurry was diluted with heptane (400 mL) and concentrated to neardryness. The solid was slurried in heptane, filtered and dried to give39.5 g (99%) of4-(3-chloro-4-fluorophenyl)-3-[4-(2-[(4-methoxybenzyl)-(methyl)amino]-ethylamino)-1,2-5-oxadiazol-3-yl]-1,2,4-oxadiazol-5(4H)-one as a tan solid.

Step 9.4-(3-Chloro-4-fluorophenyl)-3-[4-(2-(methyl)amino]ethylamino)-1,2-5-oxadiazol-3-yl]-1,2,4-oxadiazol-5(4H)-onehydrochloride

A suspension of4-(3-chloro-4-fluorophenyl)-3-[4-(2-[(4-methoxybenzyl)-(methyl)amino]-ethylamino)-1,2-5-oxadiazol-3-yl]-1,2,4-oxadiazol-5(4H)-one(39.3 g, 82.7 mmol) and NaHCO₃ (69.5 g, 827 mmol) in 1,2-dichloroethane(400 mL) was cooled to 0° C. and 1-chloroethyl chloroformate (26.6 g,20.3 mL) was added dropwise at 0-2° C. The mixture was allowed to warmto room temperature, stirred 1.25 h then heated at 40-45° C. for 2.75 h.The mixture was cooled to room temperature, filtered, and the solidswashed with DCM (500 mL). The filtrate was concentrated under reducedpressure giving a viscous brown oil. The oil was dissolved in MeOH (300mL) and stirred overnight at room temperature. To ensure completeformation of amine hydrochloride salt, 4M HCl in dioxane (20 mL) wasadded and the mixture was concentrated under reduced pressure to give aviscous brown oil. The oil was dissolved in EtOAc (150 ml). Afterstanding several minutes a precipitate began forming. When precipitationwas complete the mixture was diluted with toluene (150 ml) and the solidwas filtered. The solid was washed with 50% EtOAc/toluene (160 ml) anddried under N₂ to give 23.7 g (73%) of4-(3-chloro-4-fluorophenyl)-3-[4-(2-(methyl)amino]ethylamino)-1,2-5-oxadiazol-3-yl]-1,2,4-oxadiazol-5 (4H)-one hydrochloride asa tan solid that was used without further purification.

Step 10.4-(3-Chloro-4-fluorophenyl)-3-{4-[(2-(aminosulfonyl)(methyl)amino]ethylamino)-1,2-5-oxadiazol-3-yl}-1,2,4-oxadiazol-5(4H)-one

A solution of crude 4-(3-chloro-4-fluorophenyl)-3-[4-(2-(methyl)amino]ethylamino)-1,2-5-oxadiazol-3-yl]-1,2,4-oxadiazol-5(4H)-onehydrochloride (23.7 g, ˜60.8 mmol) and sulfamide (23.3 g, 243 mmol) inpyridine (275 mL) was heated at ˜100° C. for 2.5 h. A light suspensionformed at ˜95° C. that developed into a gummy residue at the end of thereflux period. The mixture was cooled to room temperature, the pyridinesolution decanted from the gummy residue, and the remaining pyridine wasevaporated under reduced pressure. The residual brown oil waspartitioned between EtOAc (500 mL) and 1N HCl (200 mL). The organicphase was washed sequentially with 1N HCl (2×200 mL), H₂O (200 mL), andbrine (200 mL), and then dried over Na₂SO₄, filtered and the solutionconcentrated under reduced pressure to give 31.1 g of crude4-(3-chloro-4-fluorophenyl)-3-{4-[(2-(aminosulfonyl)(methyl)amino]ethyl-amino)-1,2-5-oxadiazol-3-yl}-1,2,4-oxadiazol-5(4H)-one as aviscous brown oil that solidified on standing overnight. The crudeproduct was used without further purification.

Step 11.4-(2-[(Aminosulfonyl)(methyl)amino]ethylamino)-N-(3-Chloro-4-fluorophenyl)-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide

10% aqueous NaOH (150 mL) was added slowly to a suspension of crude4-(3-chloro-4-fluorophenyl)-3-{4-[(2-(aminosulfonyl)(methyl)amino]ethylamino)-1,2-5-oxadiazol-3-yl}-1,2,4-oxadiazol-5(4H)-one(53.3 g) in MeOH (500 mL). The reaction temperature increased from 19 to29° C. A clear brown solution slowly developed followed by formation ofa light suspension formed after 0.25 h. After 0.75 h at room temperaturethe reaction was complete. The mixture was concentrated under reducedpressure to remove most of the MeOH and the aqueous residue was dilutedwith H2O. The mixture was cooled in an ice-bath and adjusted to pH 2with conc HCl/ice to give a gummy oil. The mixture was extracted withEtOAc (1 L). The organic solution was washed with sat'd aqueous NaHCO₃(500 mL), brine (500 mL), dried over Na₂SO₄, filtered and the solutionconcentrated under reduced pressure to give a viscous brown oil thatpartially solidified on standing several days. The crude product wasredissolved in a minimal amount of EtOAc, absorbed on to silica gel anddry loaded on a column of silica gel packed in 33% EtOAc/heptane. Thecolumn was eluted with 33% EtOAc/heptane (3 L), 50% EtOAc/heptane (4 L),and 67% EtOAc/heptane (3 L). Product fractions were concentrated until aslight turbidity developed. The mixture was slowly concentrated todryness to give an off-white solid. The solid was suspended in IPA (220mL, 6 mL/g) and heated to 75° C. (clear solution at ˜30° C.). Heptane(550 mL, 15 mL/g) was added slowly via an addition funnel at 73-75° C.When addition was complete the solution was allowed to cool slowly. Whena slight turbidity developed (˜55° C.) the mixture was seeded. Solidsbegan precipitating as the mixture slowly cooled. After cooling to roomtemperature the suspension was further cooled in an ice/brine bath andstirred at −4 to 0° for 3 h. The mixture was filtered, washing thesolids with heptane (200 mL) then dried to give 31.0 g of4-(2-[(aminosulfonyl)(methyl)amino]ethylamino)-N-(3-chloro-4-fluorophenyl)-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamideas a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 11.50 (s, 1H), 8.92 (s,1H), 7.20 (dd, J=9.1 Hz, 1H), 6.98 (dd, J=6.5, 2.7 Hz, 1H), 6.76 (s,2H), 6.73-6.68 (m, 1H), 6.23 (t, J=6.0 Hz, 1H), 3.42-3.36 (m, 2H), 3.12(t, J=6.2 Hz, 2H), 2.66 (s, 3H). LCMS for C₁₂H₁₆ClFN₇O₄S (M+H)+:m/z=408.1; Found: 408.0. IC₅₀: 132 nM. This number is the average offive lots.

Compound Data

Human Indoleamine 2,3-Dioxygenasae (IDO) Enzyme Assay:

Human indoleamine 2,3-dioxygenasae (IDO) with an N-terminal His tag wasexpressed in E. coli and purified to homogeneity. IDO catalyzes theoxidative cleavage of the pyrrole ring of the indole nucleus oftryptophan to yield N′-formylkynurenine. The assays were performed atroom temperature as described in the literature using 95 nM IDO and 2 mMD-Trp in the presence of 20 mM ascorbate, 5 μM methylene blue and 0.2mg/mL catalase in 50 mM potassium phosphate buffer (pH 6.5). The initialreaction rates were recorded by continuously following the absorbanceincrease at 321 nm due to the formation of N′-formlylkynurenine (See:Sono, M., et al., 1980, J. Biol. Chem. 255, 1339-1345).

Select physical and biological activity data for the compounds ofExamples 1-19 are summarized in Table 4 below. IC₅₀ data are from theIDO enzyme assay described above.

TABLE 4 IDO Ex. IC₅₀ MS No. R₁ R₂ R₃ n (nM) [M + H] 1 NH₂ Br F 1 <200437.9, 439.9 2 Me Br F 1 <200 437.0, 439.0 3 NH₂ Br F 2 <100 451.8,453.9 4 Me Br F 2 <100 451.0, 453.0 5 NH₂ Cl F 1 <200 394.0 6 Me Cl F 1<200 393.0 7 NH₂ Cl F 2 <200 408.1 8 Me Cl F 2 <200 407.1 9 NH₂ CF₃ F 1<100 428.0 10 Me CF₃ F 1 <100 427.0 11 NH₂ CF₃ F 2 <100 442.0 12 Me CF₃F 2 <100 441.1 13 NH₂ CF₃ H 1 <500 410.0 14 Me CF₃ H 1 <200 409.1 15 NH₂CF₃ H 2 <200 424.0 16 Me CF₃ H 2 <200 423.1 17 Me CH₃ F 1 <500 373.1 18NH₂ CN F 1 <750 385.0 19 Me CN F 1 <500  406.0* *[M + Na]Compound Data

IDO IC₅₀ data for the compounds of Examples 20, 21, and 24 is providedbelow in Table 5.

TABLE 5 Ex. No. IDO IC₅₀ (nM) 20 <500 21 <750 24 <200

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of some embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe method described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

REFERENCES

The following references and any others listed herein, to the extentthat they provide exemplary procedural or other details supplementary tothose set forth herein, are specifically incorporated herein byreference in their entirety.

-   WO 99/29310-   WO 03/087347-   WO 2004/094409-   U.S. 2004/0234623-   U.S. 2006/0258719-   U.S. 2007/0185165-   U.S. Pat. No. 6,682,736-   U.S. Pat. No. 6,984,720-   U.S. Pat. No. 7,034,121-   U.S. Pat. No. 7,109,003-   U.S. Pat. No. 7,132,281-   U.S. Pat. No. 7,229,628-   U.S. Pat. No. 7,307,064-   U.S. Pat. No. 7,311,910-   U.S. Pat. No. 8,088,803-   U.S. patent Ser. No. 12/498,782-   Blank et al., Cancer Res., 64:1140-1145, 2004.-   Brown et al., J Immunol. 177:4521-4529, 2006.-   Daubener, et al., Adv. Exp. Med. Biol., 467: 517-24, 1999.-   Elpek et al., The Journal of Immunology, 178: 6840-6848, 2007.-   Gajewski et al., Cancer 1, 16:399-403, 2010.-   Gajewski et al., Immunol. Rev., 213:131-145, 2006.-   Grohmann, et al., Trends Immunol., 24: 242-8, 2003.-   Harlin et al., Cancer Immunol. Immunother., 55:1185-1197, 2006.-   Harlin et al., Cancer Res., 69(7):3077-85, 2009.-   Kline et al., Clin. Cancer Res. 14:3156-3167, 2008.-   Logan, et al., Immunology, 105: 478-87, 2002.-   Muller et al., Nature Med., 11: 312-9, 2005.-   Munn, et al., Curr Pharm Des, 9(3):257-64, 2003.-   Munn, et al., J Exp Med, 189(9):1363-72, 1999.-   Munn, et al., J Clin. Invest., 114(2): 280-90, 2004.-   Munn, et al., Science, 297: 1867-70, 2002.-   Posner et al., Hybridoma, 6(6):611-25, 1987-   Quezada et al., J. Clin. Invest., 116: 1935-1945, 2006.-   Taylor, et al., FASEB J., 5: 2516-22, 1991.-   Uyttenhove et al., Nature Med., 9: 1269-74, 2003.-   Wermuth, et al., Handbook of Pharmaceutical Salts: Properties, and    Use. Switzerland: Verlag Helvetica Chimica Acta, 2002.-   Wirleitner, et al., Curr. Med. Chem., 10: 1581-91, 2003.-   Zha et al., Nat. Immunol. 7:1166-1173, 2006.-   Zhang et al., Blood, 114:1545-1552, 2009.

The invention claimed is:
 1. A method of treating cancer in a subject inneed thereof, comprising administering to the subject an effectiveamount of an inhibitor of indoleamine-2,3-dioxygenase (IDO) and aninhibitor of the PD-L1/PD-1 pathway selected from the group consistingof BMS-936559, MPDL3280A, BMS-936558, MK-3475, CT-011, and MEDI47361;wherein the inhibitor of IDO is the compound:


2. The method of claim 1, wherein treating cancer is further defined asreducing the size of a tumor or inhibiting growth of a tumor.
 3. Themethod of claim 1, wherein the cancer is melanoma, cervical cancer,breast cancer, ovarian cancer, prostate cancer, testicular cancer,urothelial carcinoma, bladder cancer, non-small cell lung cancer, smallcell lung cancer, sarcoma, colorectal adenocarcinoma, gastrointestinalstromal tumors, gastroesophageal carcinoma, colorectal cancer,pancreatic cancer, kidney cancer, hepatocellular cancer, malignantmesothelioma, leukemia, lymphoma, myelodysplastic syndrome, multiplemyeloma, transitional cell carcinoma, neuroblastoma, plasma cellneoplasms, Wilm's tumor, or hepatocellular carcinoma.
 4. The method ofclaim 3, wherein the cancer is melanoma.
 5. The method of claim 1,wherein the inhibitors are administered to the subject at least two,three, four, five, six, seven, eight, nine or ten times.
 6. The methodof claim 1, wherein said subject is further administered a distinctcancer therapy selected from the group consisting of surgery,radiotherapy, chemotherapy, toxin therapy, immunotherapy, cryotherapyand gene therapy.
 7. The method of claim 1, wherein the cancer is achemotherapy-resistant or radio-resistant cancer.
 8. The method of claim1, wherein the subject is administered at least about 0.01, 0.02, 0.03,0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,0.8, 0.9, 1.0, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80,90, 100, 150, 200, 250, or 300 μg/kg or mg/kg of either of theinhibitors of claim 1.